Received: 8 November 2019 

|  Revised: 10 March 2020 

|
  Accepted: 14 March 2020

DOI: 10.1111/zsc.12421

ORIGINAL ARTICLE

Species delineation in the speciation grey zone—The case of

Diopatra (Annelida, Onuphidae)

Asmaa H. Elgetany1,2   | Hendré van Rensburg3  | Martin Hektoen4  |

Conrad Matthee3   | Nataliya Budaeva5  | Carol A Simon3  | Torsten H. Struck2

1
Zoology Department, Faculty of Science,
Damietta University, New Damietta,
Abstract
Central Zone, Egypt Taxonomy based on morphology can be difficult. The challenges arise from dif-
2
Natural History Museum, University of ferent sources such as poor original descriptions, new records based on inadequate
Oslo, Oslo, Norway
knowledge, uncritical application of general assumptions or presence of complexes
3
Department of Botany and Zoology,
of cryptic species. One example of problematic taxonomy is the genus Diopatra
Stellenbosch University, Stellenbosch,
South Africa Audouin & Milne Edwards, 1833 (Onuphidae, Annelida) and within it the two spe-
4
NTNU University Museum, Norwegian cies Diopatra aciculata and D. neapolitana. The species exhibit great similarity be-
University of Science and Technology, tween them casting doubts on their validity as separate species. Our study aims to
Trondheim, Norway
5
investigate whether D. aciculata and D. neapolitana should be synonymized, using
Department of Natural History, University
Museum of Bergen, University of Bergen, an integrative taxonomic approach. Therefore, we assessed 22 morphological char-
Bergen, Norway acters of 70 specimens including one specimen, which might have been erroneously
assigned to D. dentata. Additionally, sequence information of five different molecu-
Correspondence
Asmaa H. Elgetany, Zoology Department, lar markers (i.e., 16S rDNA, COI, 28S, ITS1 and ITS2) was gathered to delineate
Faculty of Science, Damietta University, possible species boundaries between these two species. Our results show some evi-
New Damietta, Central zone, 34517, Egypt.
Emails: asmaa_haris222@yahoo.com,
dence for delineating the two species, but they are not conclusive due to both pres-
Asmaa_Haris@du.edu.eg ence of shared morphological characters and conflicting evidence in the molecular
data. Accordingly, our results neither confirm nor disprove complete speciation and
Funding information
National Research Foundation both species seem to be in the grey zone of speciation. In conclusion, considering
taxonomic stability and slight support by morphological characters, we still regard
each as two independent species.

KEYWORDS
Diopatra aciculata, Diopatra dentata, Diopatra neapolitana, Onuphidae, speciation

1  |   IN T RO D U C T ION challenging, especially in annelids where cryptic diversity is

Morphology-based taxonomy including the identification and
delineation of annelid species can be extremely difficult or
even impossible (Rouse & Pleijel, 2001). For one, this stems
from epistemological problems in the process of species delin-
eation, which resulted in a plethora of species concepts (De-
Queiroz, 2007; Hausdorf, 2011; Pante et al., 2015). Despite
the uncertainties, the biological species concept (Mayr, 1942)
assuming reproductive isolation is still one of the most influ-
ential concepts. However, to assess reproductive isolation is
rife (Nygren, 2013; Nygren et al., 2018). Applying traditional
morphological species concepts, which define species as “the
smallest groups that are consistently and persistently distinct,
and distinguishable by ordinary means” (Cronquist,  1978)
is thus not a feasible taxonomic scheme for all annelids.
Genetic differences in both sympatry (Styan, McCluskey,
Sun, & Kupriyanova,  2017) and allopatry (Manchenko
& Radashevsky,  1994) can significantly enhance our un-
derstanding of reproductive isolation among taxa and can
sometimes provide evidence of reproductive isolation (Halt,

Zoologica Scripta. 2020;00:1–19. wileyonlinelibrary.com/journal/zsc © 2020 Royal Swedish Academy of Sciences     1 |

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2       ELGETANY et al.
Kupriyanova, Cooper, & Rouse, 2009; Styan, Kupriyanova,
& Havenhand, 2008). Alternative ideas such as phylogenetic
(Cracraft,  1989) or genetic (Baker & Bradley,  2006) spe-
cies concepts stress either the recognition of monophyletic
groups or that genetic isolation based on sequence variation
should instead be used as indications of species boundaries.
However, all approaches relying on sequence data might be
hampered by taking stronger population structure as indic-
ative of species boundaries (Sukumaran & Knowles, 2017).
Hence, the epistemological problems of species delineation
remain, making it difficult to delineate species on clear ob-
jective criteria.
The challenges in annelid taxonomy might be related not
only to general problems in species delineation, but also to
several practical factors (Hutchings & Kupriyanova,  2018).
The original description of the species, especially of species
described in the 18th and 19th centuries, can be poor, com-
prising only a few characters that are no longer diagnostic.
New records could be based on material collected distant
from the type locality and will then solely rely on identifi-
cation keys not developed for the collection area. In addi-
tion, annelid taxonomy was influenced by flawed concepts
such as that marine invertebrate species exhibit a wide vari-
ation in characters and hence multiple species were often
regarded as conspecific. Another flawed concept is that
Fauvel (1953) firmly believed in cosmopolitan annelid dis-
tributions and hence applied the names of European anne-
lid species to many taxa collected from remote geographical
areas such as India. Moreover, he synonymized numerous
species without indicating whether any material was actu-
ally examined to substantiate these decisions (Hutchings &
Kupriyanova,  2018). Similarly, Day (1967) still included
many European “cosmopolitan” species in South Africa de-
spite publishing an illustrated monograph on polychaetes of
the region. Hartman (1959, 1965) also synonymized many
species without any explanation. For example, she suggested
that 11 of the 66 Marphysa species should be regarded as M.
sanguinea and eight of the 22 Terebellides species as T. stro-
emii. Often, poor sampling techniques resulted in degraded
or damaged specimens which can also hamper morphological
identifications. Taxonomists also often based their work on
preserved material and not living animals, thereby neglecting
habitat types, ecology and reproductive biology (Hutchings
& Kupriyanova, 2018). Hence, conservative views in annelid
taxonomy resulted in the proliferation of perceived cosmopol-
itan species, but which often lacked strong taxonomic support
(Hutchings & Kupriyanova, 2018). Finally, understanding of
distributions of annelid species markedly changed after the
inclusion of molecular data in taxonomy. Consequently, many
species are now recognized as being complexes of cryptic
species (Bickford et al., 2007; Nygren, 2013). The Diopatra
neapolitana complex provides a good example of a species
previously thought to be cosmopolitan due to conservative
taxonomic views but is now known to comprise at least five
species (see Paxton, 2016 for summary table). However, the
distinction between D. neapolitana Delle Chiaje, 1841 and
D. aciculata Knox & Cameron, 1971, is not well defined and
thus in need of revision.
Diopatra Audouin & Milne Edwards, 1833 is the larg-
est known genus of Onuphidae, represented by about 60
species worldwide (Read & Fauchald,  2019). The genus
is distributed in intertidal and shallow subtidal areas of all
major oceans, although it is better represented in warmer
waters (Paxton, 1986). Moreover, species of this genus can
reach high densities (Cunha, Hall, & Queiroga, 2005; Dagli,
Ergen, & Cinar,  2005). Accordingly, Diopatra species play
important ecological roles by constituting a food source in
the marine food web (Rangel & Santos,  2009), stabilizing
sediment (Bailey-Brock, 1984; Luckenbach, 1986), facilitat-
ing dispersal of eelgrass and algae (Harwell & Orth, 2001;
Thomsen & McGlathery, 2005) and increasing surrounding
infaunal biodiversity (Thomsen, Muth, & McGlathery, 2011;
Woodin,  1981). Some can also be bioindicators of metal
contamination, organic matter enrichment and pharma-
ceuticals in coastal environments (Carregosa et  al.,  2014;
Freitas et  al.,  2015; Freitas, Pires, Quintino, Rodrigues,
& Figueira,  2012). Furthermore, its regenerative capac-
ity is affected by abiotic factors (i.e., pH, temperature and
salinity of the sea water) and can be used as a sensitive
marker to assess the metabolic effects of imminent climate
change on marine invertebrates (Pires, Figueira, Moreira,
Soares, & Freitas,  2015). Finally, large Diopatra species
are widely used and sold as fish bait in Europe (Arias,
Paxton, & Budaeva,  2016), Asia (Saito, Kawai, Umino,
& Imabayashi,  2014), South Africa (Simon et  al.,  2019)
and Australia (where it is also farmed, Safarik, Redden, &
Schreider, 2006).
The genus Diopatra has been historically recognized by
the presence of a unique autapomorphy—spiralled bran-
chiae (Paxton,  1993, 2002). However, species of Diopatra
also demonstrate a wide range of intraspecific variability,
often lacking distinct diagnostic characters, making them
notoriously difficult to identify (Arias & Paxton,  2013;
Hartman, 1944; Paxton, 2002). Hence, Diopatra taxonomy at
the species level remains unclear and in need of a major revi-
sion (Paxton, 1986), which is in fact a mandatory step prior
to the study of species distributional range shifts (Rodrigues,
Pires, Mendo, & Quintino, 2009). Nonetheless, members of
the D. neapolitana complex can be easily recognized by the
“ventral lobe” at the lower part of the prechaetal parapodial
lobe.
The tube-building D. neapolitana is one of the earliest
known species of the genus (Delle Chiaie, 1841). However,
the original description was brief, the diagnostic characters
were generic, not specific, and the type locality was not
exactly specified (Arias et al., 2016). Diopatra neapolitana
ELGETANY et al.
has been widely reported globally (Berke et  al.,  2010;
Dagli et  al.,  2005), namely the Red Sea and the Indian
Ocean (Wehe & Fiege,  2002), the Mediterranean Sea
(Arvanitides, 2000; Dagli et al., 2005), the coast of Portugal
(Pires, Gentil, Quintino, & Rodrigues,  2012), the Pacific
Ocean (Choe,  1960) and the Atlantic Ocean (Bergamo,
Carrerette, & Nogueira, 2019; Lourido, Cacabelos, &
Troncoso, 2008). Wethey and Woodin (2008) set the north-
ern limit of D. neapolitana in France. However, Day (1967)
regarded several species to be misidentified as D. neapol-
itana and accordingly that all records of D. neapolitana
outside the Mediterranean Sea should be considered doubt-
ful. Thus, specimens reported by Choe (1960) as D. nea-
politana may be D. sugokai Izuka, 1907 (Paxton,  1993),
although a recent study confirmed the occurrence of D.
neapolitana in Brazil (Bergamo et al., 2019). By contrast,
D. aciculata from Australia is very similar to D. neapol-
itana and was in the original description delimited from
D. neapolitana by four characters: a knob-like secondary
tooth on the pseudocompound hooks; aciculae with sharply
upwardly turned distal end; a smooth tube; and lastly dor-
sal cirri extending beyond the tips of the branchiae. Later,
Paxton (1993) stated that no distinct differences could be
observed between the two species, with the reported sec-
ondary tooth on the hooks being an optical illusion, while
the shape of the aciculae is common in all large Diopatra
species. Thus, the only characters to distinguish D. acicu-
lata from D. neapolitana were its slightly longer dorsal cirri
and having a smoother tube. Nonetheless, Paxton (1993)
suggested that D. aciculata be retained as a separate taxon
until the opposite could be proven. Rodrigues et al. (2009)
seemingly confirmed the validity of both species based on
genetic differences in the mitochondrial markers COI and
16S between two specimens from Portugal and one speci-
men from Australia.
Studies on the reproductive biology of both D. neapoli-
tana from Northern Spain, Bay of Biscay, and D. aciculata
from New South Wales, Australia, showed both species to
be broadcast spawners with mature oocytes of similar size
(200–240 µm; Arias et al., 2016; Paxton, 1993). At least in D.
neapolitana, protandric hermaphroditism has been reported
with hermaphroditic specimens bearing dorsal external sper-
maducal papillae. These papillae were originally used as a
diagnostic character for D. cryptornata Fauchald, Berke, and
Woodin (2012), which was later synonymized with D. nea-
politana based on identical COI and 16S sequences (Arias
et al., 2016). Dorsal papillae are delicate minute structures re-
stricted to the hermaphrodites within the studied population
and used for sperm storage and release. They have not been
found in D. aciculata or in any other Diopatra species. Only
male and female specimens were reported for D. aciculata al-
though no detailed histological investigation was performed
(Paxton, 1993).
  
|
   3
In this study, we address the complicated taxonomic issue
of the validity of D. neapolitana and D. aciculata as separate
species. Therefore, we use both molecular (i.e., the genetic
markers 16S, COI, 28S, ITS1 and ITS2) and morphological
data (i.e., 22 characters for 70 individuals) from a substantial
part of the distribution ranges of both species to determine
whether differences between the two species were present in
both sources of data. If differences are present, validity of
both species would be supported, if not, the species should
be synonymized.
2  |  M ATERIAL AND M ETHODS
2.1  | Sampling
For this study, we collected specimens, which belonged to
either D. aciculata or D. neapolitana from different locali-
ties in the intertidal zone (Figure  1). Two specimens were
from Egypt (Suez Canal; 30°50′31.5″N 32°18′54.8″E; AE1
& NE1 in Table  S1), 29 from South Africa (Bollard Bay,
Knysna Estuary; 34°03′43″S 23°03′16″E; ASA2, ASA3
& ASA5; Knysna Estuary; 34°03′37.4″S 23°02′52.5″E;
KN_01, KN_03, KN_04, KN_05, KN_06, KN_07, KN_08,
KN_09, KN_10, KN_11, KN_12, KN_14, KN_15, KN_16
& KN_17; Swartkops Estuary, Port Elizabeth; 33°51′37.9″S
25°37′03.9″E; PE_02, PE_04, PE_06, PE_07, PE_08, PE_09,
PE_10, PE_11, PE_12, PE_17 & PE_18; in Table S1) and six
from Spain (San Simón, Vigo Estuary; 42º18′N 8º37′W; O
Grove, Arousa Estuary; 42º29′N 8º51′W; ZMBN 106619,
El Puntal, Villaviciosa Estuary; 43º31′N 5º23′W; ZMBN
106620 and ZMBN 106622; NS1-3 & CS1-3 in Table S1).
Animals were anaesthetized in a 7% MgCl2 solution. A sec-
tion of the midbody was removed and stored in 96% ethanol
for molecular analysis. The rest of the animal was fixed in
4% sea water formalin and stored in 70% ethanol for future
reference. Alternatively, specimens were entirely preserved
in ethanol. All specimens are deposited at the Iziko South
African Museum, Cape Town, the Natural History Museum
in Oslo, Department Museum of Damietta University or the
University Museum of Bergen (ZMBN).
2.2  |  Morphological characterization and
data analysis
For morphological examination, these specimens were
complemented by 12 samples of D. neapolitana includ-
ing the neotype and three paratypes from collections of the
Museo Nacional de Ciencias Naturales, Spain, and Natural
History Museum, Norway, and 46 specimens of D. acic-
ulata including the holotype from the National Museum
of Victoria, Western Australian Museum and Australian
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4       ELGETANY et al.

F I G U R E 1   Map of Diopatra species and localities used in this study (as far as they are known, see Table S1). Coloured squares indicate
different species: red—D. neapolitana (positions of D. biscayensis, D. cuprea, D. marocensis and D. micrura are not shown as they occur at similar
localities than D. neapolitana along the Portuguese, Spanish and French Atlantic coastline), yellow—D. aciculata, blue—D. dentata, turquoise—
D. sugokai, light green—D. tuberculantennata, dark green—D. ornata, pink—Dioptra sp. Map based on URL: https://www.google.com.eg/
maps/@30.84208​33,68.17459​78,14902​315m/data=!3m1!1e3

Museum, all Australia (Table S1). Additionally, one sam-
ple of D. dentata, deposited as a voucher for sequences
GQ478129 and GQ497522, was examined as it was possi-
ble that this specimen is not D. dentata, but either D. nea-
politana or D. aciculata (Paxton, pers. comm). For each
of these 70 adult specimens, 22 morphological characters
were assessed, if possible, given the quality of the material
and permission from the collections (Table S2). We meas-
ured the (1) width, (2) length of branchia and (3) length of
dorsal cirri at the 10th chaetiger in mm. To account for dif-
ferences due to size, length of the dorsal cirri was also set
in relation to both (4) length of the parapodium at the 10th
chaetiger and (5) length of the branchia at the 10th chaeti-
ger. (6) Length of the peristomial cirri was also compared
to length of the peristomium. We counted the number of
rings of the ceratophores of the (7) median and (8) lateral
antennae and (9) palps and (10) the maximum number of
whorls per branchia. We also assessed the length of the
(11) median and (12) lateral antennae and (13) palps by
determining the chaetiger to which each reached. We de-
termined the chaetiger at which the (14) first and (15) last
branchiae, (16) first and (17) last ventral lobes and (18) first
subacicular hook occurred and (19) coloration started and
(20) prechaetal lobe end. Finally, we assessed (21) if the
shape of the frontal lips was subulate attached to either side
of apex (short and thick) or distinctly separated (long and
thin) and (22) if the shape of acicular chaetae was straight,
slightly or strongly curved. All counts, measurements and
colour descriptions are based on preserved samples.
The data were analysed using RStudio Version 1.1.463
(http://www.rstud​ io.com/) using the libraries FactoMineR,
missMDA and psych. We conducted different principal com-
ponent analyses (PCA) applying different strategies deal-
ing with missing data in the data set. First, we excluded all
specimens with missing data by applying the na.omit option.
Second, we calculated the value of the missing data point as
the average of the other data for that character. Third, we im-
puted the data point using FactoMineR by estimating the num-
ber of relevant components using the k-fold cross-validation
method. Imputing the missing data instead of averaging has
the advantage that imputed missing values have no effect on
the PCA as the process considers the similarities between the
observations and the relationship between variables. Hence,
the PCA is performed with only the observed values, but al-
lows inclusion of all specimens. As the missing data nonethe-
less generate uncertainty in the position of a specimen, we also
included an assessment of the error margins in the imputation
process by using MIPCA with 1,000 bootstrap replicates.
For each PCA, we determined the proportional and cu-
mulative proportional vicariance, the values for the variables
in relation to the PCs, contributions of the variables to each
PC and summary statistics including the new averaged val-
ues for the data table. We also generated hierarchical clusters
based on FactoMineR. Additionally, we calculated the mul-
tidimensional morphological disparity (MMD) indices based
on all PCs, which explained 95% of the variability in the data
set (Struck, Koczula, Stateczny, Meyer, & Purschke, 2017).
In brief, the coordinate for each specimen and PC was
ELGETANY et al.
determined and the Euclidean distance (i.e., the MMD index)
for each specimen pair was calculated. Hence, the MMD
index is a pairwise distance of morphological disparity
across several dimensions of the morphospace and is anal-
ogous to genetic pairwise distances. Based on these MMD
indices, a hierarchical clustering was conducted as well as
box plots generated. Additionally, Student's t test and Tukey's
HSD tests were conducted to test if the interspecific pairwise
distances were significantly larger than the intraspecific ones.
Finally, we analysed each character individually by cal-
culating summary statistics and box plots for both D. acicu-
lata and D. neapolitana. We also conducted statistical tests
between the species to test if observed differences were sig-
nificant. Therefore, we checked if distribution of each char-
acter in each species was normal using a Shapiro–Wilk's W
test for normality. For the continuous characters, we only
conducted a Student's t test when data were normal for both
species; otherwise, a Mann–Whitney U test was applied. For
the two categorical characters (#21 & #22 above), a Χ2 test
was performed. The code used in R studio can be found in
Supplementary Material 1.
2.3  |  Molecular analysis
Molecular data were generated at two laboratories, and there-
fore, different protocols had been applied. We used five dif-
ferent markers, in particular, the nuclear fragments comprising
28S rRNA, ITS1 and ITS2 and the mitochondrial 16S rRNA
and COI. Genomic DNA was extracted using the DNeasy
Tissue Kit (Qiagen) or the Quick-DNA miniprep plus kit
(Zymo Research) according to manufacturers’ instructions
with at least two elution steps to increase the amount of DNA.
The genes were amplified using either the Multiplex PCR Kit
(Qiagen) in a 20 μl reaction or EconoTaq® PLUS GREEN 2X
Master Mix (Lucigen) in a 25 μl reaction. The details of each
PCR are given in Table S3. PCR fragments of the expected sizes
were purified enzymatically using ExoProStarTM (Qiagen).
Both strands were sequenced using Sanger sequencing either
at Macrogen Inc. (South Korea) or at the Central Analytical
Facility of Stellenbosch University. Sequences were assembled
into contigs using CodonCode Aligner version 6.0.2.
In addition to the new sequences, we also obtained pub-
licly available sequences of D. aciculata, D. neapolitana and
the dubious D. dentata and of other Diopatra or onuphid
species as outgroup taxa (Table  S1). The data set of each
gene was aligned individually using MAFFT and the au-
tomatically chosen L-INSI option (Katoh, Kuma, Toh, &
Miyata, 2005). Maximum Likelihood (ML) analyses of each
gene individually were conducted using IQ-TREE multicore
version 1.5.5 (Nguyen, Schmidt, Haeseler, & Minh,  2015)
with 1,000 bootstrap (BS) replicates, 300 initial parsimony
trees, 15 trees to be maintained in the candidate set during
  
|
   5
ML tree search and the automatic model selection option
MFP  +  LMSS. Additionally, haplotype networks of each
gene comprising only specimens of D. aciculata, D. neapoli-
tana and D. dentata were built using TCS (Clement, Posada,
& Crandall, 2000) with gaps treated as fifth state and a con-
nection limit of 90%. The networks were further processed
with tcsBU (Murias Dos Santos, Cabezas, Tavares, Xavier,
& Branco, 2015). Finally, to delineate putative species in our
data sets, we used the Automatic Barcode Gap Discovery
(ABGD) (Puillandre, Lambert, Brouillet, & Achaz,  2012)
method as implemented at http://wwwabi.snv.jussi​eu.fr/publi​
c/abgd/abgdw​eb.html with different values for the relative
gap width X (i.e., 0.005, 0.01 and 0.05) and default settings.
3  |  RESULTS
3.1  |  Molecular data
The COI data set comprises 29 specimens from South Africa,
one from Australia, two from Egypt, two from India, two
from Brazil and 33 from Spain, Portugal or France and the
sequence of Diopatra dentata and has a total of 553 posi-
tions. In the phylogenetic analysis, monophyly of a clade
comprising all specimens of D. aciculata and D. neapolitana
plus the one specimen of D. dentata is maximally supported
by a BS value of 100 (Figure 2a). Within this clade, a clade
comprising all specimens from Australia and South Africa,
which are supposedly D. aciculata, plus D. dentata from
Australia and the specimen AE1 from Egypt is also strongly
supported (BS = 99, Figure 2a). This clade is nested within
all other supposed D. neapolitana from France, Portugal,
Spain, Brazil and India, plus the specimen NE2 from Egypt.
However, support for this placement is weak. In the haplo-
type network reconstruction, two separate networks were re-
covered, which are not connected to each other, as they are
more than 14 substitutions, the connection threshold, differ-
ent from each other (Figure 3a). One of the two contains all
specimens from Australia (including D. dentata) and South
Africa as well as AE1 from Egypt. The other comprises all
remaining specimens that are supposedly D. neapolitana,
plus NE2 from Egypt. The results of the ABGD are the
same across the different settings and minimally recover two
groups. The maximal intraspecific pairwise genetic distance
p within each group was <.0359. Again, one group comprises
the specimens from Australia (including D. dentata), South
Africa and AE1, while the other includes all others including
NE2 from Egypt.
The data set of the 16S fragment consists of 520 positions
and three specimens from South Africa, one from Australia,
eight from Spain, Portugal or France, two from Egypt and
one of D. dentata from Australia. The phylogenetic recon-
struction finds again strongly supported monophyly of a
 |
6       ELGETANY et al.

D. aciculata (KN_14)
D. aciculata (ASA3)
(a) D. aciculata (KN_11) (c)
D. aciculata (KN_06) D. aciculata (ASA2)
D. aciculata (KN_12) 100
Legend D. aciculata (PE_04) D. neapolitana (NS2)
D. aciculata (KN_17) D. aciculata (ASA5)
D. aciculata D. dentata (Dvou)
D. aciculata (ASA2) D. neapolit ana (CS3)
Australia D. aciculata (KN_04) D. aciculata (AE1)
D. aciculata (PE_18)
South Africa D. aciculata (KN_09) D. neapolitan a (NE2)
Egypt D. aciculata (PE_02)
D. neapolitana (CS2)
D. aciculata (AE1)
D. neapolitana D. aciculata (PE_10) 99 Onuphi s iride scens
D. aciculata (PE_09)
D. aciculata (PE_11) Onuphis elegans
Egypt D. aciculata (KN_03) Hyalinoecia tubicola
Spain/Portugal/France D. aciculata (PE_07) 0.02
D. aciculata (PE_08)
India/Unknown D. aciculata (MOL30)
99 D. aciculata (KN_10)
Brazil D. aciculata (KN_08)
D. aciculata (PE_06)
D. aciculata (KN_15)
D. aciculata (PE_17)
D. acciulata (KN_05)
D. aciculata (PE_12)
D. aciculata (KN_01)
D. aciculata (KN_16)
D. aciculata (KN_07)
D. aciculata (ASA5)
D. aciculata (ASA3)
D. neapolitana (MOL26)
D. neapolitana (MOL25)
D. neapolitan a (MOL17) D. neapolitana (CS3)
D. neapolitan a (MOL10) (b)
D. neapolitana (NS2)
D. neapolit ana (MOL11)
D. neapolitan a (MOL19) D. neapolitana (CS1)
D. neap olitan a (MOL14) D. neapolitan a (NE2)
D. neap olitan a (MOL2) D. neapolitana (MOL2)
D. neapolitan a (MOL9) D. neapolitana (CS2)
D. neapolita na (MOL27) D. neapolitana (MOL1)
D. neapolitana (MOL28) 95 D. aciculata (ASA2)
D. neapolita na (MOL23) D. dentata (Dvou)
D. neapolita na (CS3) D. aciculata (AE1)
D. neap olitan a (CS2)
D. neapolit ana (NS1) D. aciculata (ASA3)
D. neapolitan a (NE2) D. aciculata (ASA5)
D. neapolitan a (MOL4) D. aciculata (MOL30)
D. neapolitana (MOL6) D. sugokai (MOL60)
D. ne apolitan a (MOL5) Diopatra sp. (MOL58)
D. neapolitan a (MOL12) 100 D. biscayensis (MOL31)
D. neapolitan a (MOL21) 97 D. sugokai (MOL61)
0.05
D. neapolitan a (MOL65)
D. neapolitan a (MOL8) 99 100 Diopatra cf. ornata
D. neapolitan a (NS2) Diopatra ornata
100 D. neapolitana (MOL16) 100 D. tuberculantennata (MOL62)
D. neap olitan a (CS1) D. tuberculantennata (MOL63)
D. neapolitana (MOL24) D. marocensis (MOL49)
D. neap olitan a (MOL29) Diopatra sp. (MOL59)
D. neap olitan a (MOL20) Diopatra sp. (MOL57)
D. neap olitan a (MOL7) D. micrura (MOL55)
D. neap olitan a (MOL1)
D. neapolitan a (MOL15) 0.07
D. neapolit ana (MOL18)
D. neapolitan a (MOL64)
D. neapolita na (MOL3) Amer iconuph is reesei
D. neap olitan a (MOL13) (d)
D. neapolita na (MOL22) D. aciculata (AE1)
D. biscayensis (MOL33) D. neapolitan a (NE2)
D. biscayensis (MOL31)
D. biscayensis (MOL39) D. aciculata (ASA2)
D. biscayensis (MOL41) D. neapolitan a (CS2)
D. biscayensis (MOL35)
D. aciculata (ASA5)
D. biscayensis (MOL44)
D. biscayensis (MOL32) D. aciculata (ASA3)
0.06
D. biscayensis (MOL37) D. neapolit ana (CS3)
D. biscayensis (MOL36)
D. biscayensis (MOL40)
D. biscayensis (MOL38) Amer iconuphis reesei
100 D. biscayensis (MOL42) (e)
D. biscayensis (MOL43) D. neapolitan a (NE2)
99 D. biscayensis (MOL34)
D. cuprea (MOL45) D. aciculata (AE1)
100 D. cuprea (MOL48) 97
100 D. neapolit ana (CS2)
D. cuprea (MOL46)
D. cuprea (MOL47) D. neapolit ana (CS3)
D. marocensis (MOL50)
D. marocensis (MOL49) 100 D. aciculata (ASA5)
100 D. marocensis (MOL54)
D. marocensis (MOL52) D. aciculata (ASA2)
D. marocensis (MOL53) 0.03
96 D. marocensis (MOL51) D. aciculata (ASA3)
100 D. micrura (MOL55)
D. micrura (MOL56)
ELGETANY et al.
F I G U R E 2   Maximum likelihood phylogeny of each gene analysed in this study: (a) COI, (b) 28S, (c) 16S, (d) ITS1 and (e) ITS2. Bootstrap
support ≥ 95 is given above every branch. Specimens of D. neapolitana and D. aciculata are coloured based on species and regions. Specimen ID
as in Table S1 is provided after the species name
F I G U R E 3   TCS networks of each
(a)
gene analysed in this study: (a) COI, (b)
16S, (c) ITS1 and (d) ITS2. Haplotype
networks are coloured based on species
and regions. Uncoloured dots represent
hypothetical haplotypes. Size of coloured
dots corresponds to number of specimens
Legend
D. aciculata
Australia
South Africa
Egypt
D. neapolitana
Egypt
Spain/Portugal/France
India/Unknown
Brazil
clade comprising these specimens (BS = 95, Figure 2b). In
addition, within this clade, one comprising all supposed D.
aciculata from South Africa and Australia, plus AE1 from
Egypt and Diopatra dentata from Australia is again recov-
ered, but support is weak. Similarly, monophyly of all sup-
posed D. neapolitana from Spain, Portugal or France, plus
NE2 from Egypt is also recovered, but with low support. The
network reconstruction finds a single network, in which these
two groups are separated by two substitutions (Figure 3b). All
specimens from Spain, Portugal and France plus NE2 share
the same 16S haplotype. The specimens from Australia,
South Africa and AE1 exhibit three haplotypes. AE1 shares
a haplotype with two specimens from South Africa, and one
specimen each from Australia (i.e., D. dentata) and South
Africa also shares a haplotype. The ABGD results also find
minimally two groups across the different settings, which
correspond to the two clades of the phylogenetic reconstruc-
tion. The maximal intraspecific pairwise genetic distance p
within each group of <.00028 is much lower than for the COI
data set.
For the 28S gene comprising 632 positions, all spec-
imens (three each from South Africa and Spain, and two
from Egypt) display identical nucleotide sequences and are
one haplotype (Figure 2c). Accordingly, network and ABGD
analyses cannot be conducted.
Similarly, the ITS1 gene comprising 491 positions also ex-
hibits very little differences among all specimens (three from
  
   7
|
(b)
(c)
(d)
South Africa, and two each from Spain and Egypt). Accordingly,
the relationships between the specimens cannot be resolved
with strong nodal support and the specimens, which are sup-
posedly D. aciculata or D. neapolitana, are nested among each
other (Figure  2d). The network analyses reveal some differ-
entiation between them (Figure 3c). The specimen AE2 from
Egypt is three substitutions different from the haplotype shared
by two specimens from Spain, while the specimen NE2 is five
substitutions different from two specimens from South Africa.
All specimens from South Africa are only one substitution dif-
ferent from those from Spain. Accordingly, the ABGD does not
find groupings corresponding to the results of the mitochon-
drial genes. Again, across all settings minimally two groups are
found with p <.0046. One group comprises only the specimen
NE2 and the other one all remaining specimens.
The data set of the ITS2 gene with two specimens each
from Spain and Egypt and three from South Africa and 419
positions exhibits more variability. In the phylogenetic recon-
struction, the two specimens CS2 and CS3 from Spain group
together with a BS value of 100 (Figure 2e). These two spec-
imens are nested among the three specimens (i.e., ASA2, 3 &
5) from South Africa, and this clade also obtains a BS value of
100. The ITS2 sequences of the two specimens AE1 and NE2
from Egypt are identical (Figures 2e, 3d). The network analy-
sis also shows that the two specimens CS2 and CS3 are quite
different from the other specimens by at least five substitutions.
The specimens AE1 and NE2 from Egypt are two substitutions
 |
8      

T A B L E 1   Statistical measurements for the different characters for each species

D. aciculata D. neapolitana

Number in text Character n Median Mean Mode(s) SD Min Max n Median Mean Mode(S) SD Min Max
1 Width 10th chaetiger 51 5.5 5.5 NA 0.9 3.5 7 19 6 5.92 NA 0.82 4 7
7 Median antenna ceratophore 50 14 NA 12 & 14 1.46 11 17 17 12 NA 12 1.41 7 12
11 Length of median antenna 42 10 9.43 NA 2.76 5 15 15 7 7.07 NA 1.75 4 11
8 Lateral antenna ceratophore 51 15 NA 15 1.38 13 19 18 14 NA 14 1.9 8 16
12 Length of lateral antennae 45 9 9.96 NA 2.53 5 16 17 8 7.53 NA 1.94 3 10
9 Palp ceratophore 51 14 NA 14 1.26 11 16 19 12 NA 12 & 14 2.2 7 15
13 Length of palp 42 3 3.45 NA 1.47 1 8 17 2 2.35 NA 0.7 1 4
21 Frontal lips 37 2 NA 2 0.43 1 2 17 1 NA 1 0.24 1 2
6 Peristomial cirri length 43 2 1.73 NA 0.33 1 2 18 1.5 1.6 NA 0.61 0.3 3
14 Branchia first chaetiger 51 4 NA 4 0.6 3 6 19 4 NA 4 0.61 4 6
15 Branchia last chaetiger 39 51 50.1 54 5.72 41 64 18 52 NA 51 & 52 7.01 38 69
10 Branchia whorls 50 15.5 NA 14 & 17 2.25 10 20 19 15 NA 14 & 17 1.84 12 18
2 Branchia length at 10th chaetiger 51 4.5 4.59 NA 0.85 3 7 19 5 5.05 NA 1.12 3 7.5
19 Coloration appears 44 5 NA 6 1.73 1 7 19 5 NA 1 4.15 1 14
16 Ventral lobe first chaetiger 50 5 NA 5 0.83 4 8 18 5 NA 5 0.69 4 6
17 Ventral lobe last chaetiger 48 24 NA 18 6.52 16 43 18 28 NA 27 & 28 10.63 19 67
3 Dorsal cirri length 10th chaetiger 51 3.25 3.21 NA 0.66 1.5 4.75 19 2.5 2.39 NA 0.59 1.5 3.5
4 Dorsal cirri to parapodium 51 3 2.97 NA 0.52 2 5 18 2 2.08 NA 0.31 1.5 3
5 Dorsal cirri to branchiae 50 0.75 0.75 NA 0.09 0.5 0.9 18 0.3 0.35 NA 0.1 0.3 0.6
20 Prechaetal lobe last chaetiger 48 19 NA 25 5.2 10 29 18 24 NA 24 & 26 4.53 15 30
18 Subacicular hook first chaetiger 49 16 NA 17 NA 11 22 19 21 NA 17 10.71 17 52
22 Acicular chaetae 46 1 NA 1 NA 1 3 16 1 NA 1 0.4 1 2
Note: NA, not applicable; n, number of specimens; SD, standard deviation; min, minimal value; max, maximal value.
ELGETANY et al.
ELGETANY et al.
different from the South African haplotype. The ABGD analy-
ses find across all settings minimally only a single group with
p <.0077. At a p <.0046, three groups are retrieved: one group
containing CS2, the second CS3 and the third all others. Hence,
the results again do not reflect the splitting found in the mito-
chondrial data.
3.2  |  Morphological data
For the morphological analyses, all specimens from Australia
and South Africa were considered as being D. aciculata
(Paxton,  1993; van Rensburg,  2019 and van Rensburg et  al.,
unpublished data). The voucher specimen for the supposed D.
dentata sequence and the specimen AE1 from Egypt were also
assigned to D. aciculata given the results from the mitochon-
drial data. In contrast, all specimens from France, Portugal and
Spain were treated as D. neapolitana (Arias et al., 2016) and
the specimen NE2 from Egypt was also included. For each
character and species, the median and mean or mode and the
standard deviation and range are determined (Table  1). Nine
characters exhibit highly significant differences between the
two species (p <.001, Table 2) and three additional ones with
significant differences (p <.01, Table 2). These 12 characters
are number of rings at the ceratophores and the lengths of the
median and lateral antennae and palps, shape of frontal lips,
last chaetiger with a ventral lobe, length of the dorsal cirri at
the 10th chaetiger in mm and in relation to branchia and para-
podium at the same chaetiger and the first chaetiger with sub-
acicular hooks. However, even though nine of these characters
are highly significant, it does not mean that the distributions of
these characters are not overlapping (Figure 4). Not consider-
ing outliers, all these characters overlap except for the shape
of the frontal lips, and lengths of the dorsal cirri in relation to
both the branchiae and the parapodium at the 10th chaetiger.
Considering only an overlap of the quartile borders, the two
species overlap in the length of the lateral antennae, and the
number of rings at the ceratophore of both the median antenna
and palp. We also checked if the characters correlate with body
size as gauged by body width at the 10th chaetiger within each
species. However, there are only very rarely strong correlations
between each of the characters and body width in each species.
Of the significant characters, only the length of the dorsal cirrus
shows a weak positive correlation in both D. neapolitana and
D. aciculata (i.e., R2 = .25 and .30, respectively) and the last
chaetiger with subacicular hooks in D. neapolitana (R2 = .12).
Of the non-significant characters, strong correlations are ex-
hibited in the length of the branchia at the 10th chaetiger (D.
neapolitana R2 = .16 and D. aciculata R2 = .63), the last chaeti-
ger with branchiae (D. neapolitana R2 = .16 and D. aciculata
R2 = .32) and the last chaetiger with prechaetal lobes (D. nea-
politana R2 = .20 and D. aciculata R2 = .21). Interestingly, the
last chaetiger with prechaetal lobes is negatively correlated in
  
|
   9
D. neapolitana. Additionally, the last chaetiger with a ventral
lobe is correlated with body size in D. neapolitana (R2 = .12)
and maximum number of whorls at branchiae in D. aciculata
(R2 = .21). All other characters have R2 values below .10.
To assess the differences between the two species with-
out assigning them to the two species first, we conducted
different PCAs. Excluding all specimens with missing data
resulted in a data set of only 29 specimens. In this analysis,
the first principal component (PC1) explains 30.94% of the
variance in the data set and the second 12.44% (Figure 5a).
The strongest contributions to PC1 have the ratio of the
lengths of the dorsal cirrus and the branchiae or the para-
podium at the 10th chaetiger, the shape of the frontal lip,
and the lengths and number of rings of the ceratophore of
both the median and lateral antennae contributing more
than 50% to the load. For the second component (PC2),
the strongest contribution by far (i.e., 24.30%) comes from
the length of the dorsal cirrus at the 10th chaetiger. The
specimens previously assigned to D. neapolitana and the
ones assigned to D. aciculata are clearly separated by PC1.
We also calculated the MMD indices (pairwise distances
of morphological disparity across several dimensions of
the morphospace) from these results. The first 13 princi-
pal components together explained 95% of the variance
in the data sets and hence were used to calculate the in-
dices. Comparing the MMD indices within and between
the two species showed that interspecific MMD indices
are generally higher than intraspecific ones of each species
(Figure 5b). Both Student's t test and Tukey's HSD showed
that the interspecific MMD indices are highly significantly
different from the intraspecific indices of each species (all
p  <.001). Nonetheless, based on the results of the PCA,
we also applied two hierarchical clustering approaches
without assigning specimens to species. One approach was
implemented in FactoMineR and is based on the two first
principal components. The dendrogram generated from
this clustering shows that D. aciculata is paraphyletic with
respect to D. neapolitana (Figure 6a). On the other hand,
the specimen NS2 of D. neapolitana is placed among spec-
imens of D. aciculata. The second approach used the MMD
indices and although different in many aspects also finds
these two results (Figure 6b).
In the next PCA analysis, the values of the missing data
were calculated as the average for that character to include
all specimens. The first principal component (PC1) explains
only 23.92% of the variance in this case and the second
(PC2) still 12.78% (Figure  5c). For both components, the
same characters as above contribute the strongest. However,
the specimens previously assigned to D. neapolitana and
to D. aciculata, respectively, are not as clearly separated
by PC1. Even considering both components together, the
specimens AAMS40, which is supposedly D. aciculata,
Dvou, supposedly D. dentata, and CS2, supposedly D.
 |
10       ELGETANY et al.

T A B L E 2   Results of the statistical significance of the observed difference between the two species

Shapiro–Wilk's W test for

normality Significance tests

D. aciculata D. neapolitana t Test Mann–Whitney U Χ2 test

Number in
manuscript Character W p W p p W p Χ2 df p
1 Width 10th chaetiger 0.9561 .0569 0.9042 .0580 .0790 NA NA NA NA NA
7 Median antenna 0.9319 .0065 0.7490 .0004 NA 79.5 .0000 NA NA NA
ceratophore
11 Length of median antenna 0.9514 .0726 0.9571 .6422 .0031 NA NA NA NA NA
8 Lateral antenna 0.9248 .0032 0.8548 .0101 NA 151.5 .0000 NA NA NA
ceratophore
12 Length of lateral antennae 0.9672 .2275 0.9111 .1045 .0007 NA NA NA NA NA
9 Palp ceratophore 0.9132 .0012 0.8947 .0391 NA 217.5 .0003 NA NA NA
13 Length of palp 0.8844 .0005 0.8144 .0032 NA 183 0.0024 NA NA NA
21 Frontal lips 0.5338 .0000 0.2622 .0000 NA NA NA 20.1 1 .0000
6 Peristomial cirri length 0.7261 .0000 0.9105 .0879 NA 325.5 .2901 NA NA NA
14 Branchia first chaetiger 0.7553 .0000 0.6848 .0000 NA 566.5 .1980 NA NA NA
15 Branchia last chaetiger 0.9409 .0407 0.9044 .0687 NA 433 .1606 NA NA NA
10 Branchia whorls 0.9625 .1134 0.9349 .2132 .6400 NA NA NA NA NA
2 Branchia length at 10th 0.9449 .0193 0.9694 .7643 NA 610.5 .0930 NA NA NA
chaetiger
19 Coloration appears 0.8866 .0004 0.8941 .0380 NA 452.5 .6049 NA NA NA
16 Ventral lobe first chaetiger 0.8567 .0000 0.8083 .0020 NA 339 .0955 NA NA NA
17 Ventral lobe last chaetiger 0.8584 .0000 0.7076 .0001 NA 657.5 .0011 NA NA NA
3 Dorsal cirri length 10th 0.9673 .1697 0.9086 .0696 .0000 NA NA NA NA NA
chaetiger
4 Dorsal cirri to parapodium 0.7272 .0000 0.6439 .0000 NA 71.5 .0000 NA NA NA
5 Dorsal cirri to branchiae 0.9251 .0036 0.5895 .0000 NA 5 .0000 NA NA NA
20 Prechaetal lobe last 0.9446 .0246 0.9360 .2473 NA 584 .0287 NA NA NA
chaetiger
18 Subacicular hook first 0.9603 .0975 0.7018 .0001 NA 830 .0000 NA NA NA
chaetiger
22 Acicular chaetae 0.4716 .0000 0.4843 .0000 NA NA NA 2.4569 2 .2928
2
Note: W & X  = test value; df = degree of freedom; p = probability. Bold values: p <.001; italic values: p <.01.

neapolitana overlap. The specimens from South Africa are
clearly grouped with the specimens from Australia, which
have been previously assigned to D. aciculata. However,
the two specimens from Egypt are slightly set apart from
the other specimens and both overlap more with the D.
aciculata specimens than the D. neapolitana ones in PC1,
while in PC2 they are at the edge of both. Accordingly, the
distributions of the intraspecific and interspecific MMD
indices, which are based on the first 16 principal compo-
nents, are not so strongly separated from each other as be-
fore, especially the intraspecific values of D. neapolitana
(Figure 5d). Nonetheless, both Student's t test and Tukey's
HSD still reveal highly significant differences between the
inter- and intraspecific values of each species (all p <.001).
The clustering analysis based on the first two components
finds both species monophyletic as previously assigned
(Fig. S1a), except that AAMS40 is placed in D. neapoli-
tana and NE2 in D. aciculata. The clustering analysis based
on 16 components finds D. neapolitana paraphyletic with
respect to D. aciculata with NE2 and NPara1 placed within
D. aciculata (Fig. S1b).
Finally, instead of averaging we imputed the missing
data. The first principal component (PC1) explains 24.77%
of the variance and the second (PC2) 13.35% (Figure 5e). As
before, the same characters contribute the strongest to each
component. However, the specimens previously assigned to
ELGETANY et al.   
|
   11

D. neapolitana and D. aciculata, respectively, are again not 4  |  DISCUSSION

as clearly separated either by PC1 or by PC1 and PC2 to-
gether. When including the uncertainty due to the missing
data, the overlap is much bigger than in the previous analy-
ses and now includes the specimens AAMS2, AAMS10 and
AAMS40, which are supposedly D. aciculata, Dvou, sup-
posedly D. dentata, CS2 and NPara3, supposedly D. nea-
politana, and both from Egypt. As above, the South African
specimens group together with the D. aciculata specimens
from Australia. The overlap of the distributions of the intra-
specific and interspecific MMD indices is similar to the one
above, which are based on the first 15 principal components
(Figure 5f). Nonetheless, both Student's t test and Tukey's
HSD again show highly significant differences between the
interspecific and intraspecific values of each species (all
p <.001). Moreover, both clustering analyses find D. nea-
politana and D. aciculata monophyletic, except that either
NPara1 and NE1 or NPara1, NE1, CS1, CS2 and CS3 are
placed in D. aciculata (Figure 6c, d).
Although our study conducted the most thorough analyses
so far concerning both molecular and morphological data, it
could not provide support for separating the two Diopatra
species unequivocally. Thus, whether D. aciculata and D.
neapolitana should be treated as a single or separate species
remains unresolved.
Of the 22 morphological characters examined, nine
exhibited highly significant differences between the two
species if specimens were assigned to D. aciculata or
D. neapolitana in accordance with either mitochondrial
DNA data or locality (i.e., Australian and South African
specimens to D. aciculata and European specimens to D.
neapolitana). Similarly, variation in MMD indices is sig-
nificantly higher between than within species. Hence, there
are seemingly sufficient differences in morphological char-
acters to differentiate between species. However, the char-
acters, which differ significantly, might not be independent

Shape of frontal lips Number of rings of the ceratophore Length of lateral antennae
long & thin

of the lateral antenna until chaetiger number

8 10 12 14 16
10 12 14 16 18
short & thick

6
4
8

D. aciculata D. neapolitana D. aciculata D. neapolitana D. aciculata D. neapolitana

First chaetiger with subacicular hooks Number of rings of the ceratophore Number of rings of the ceratophore
of the median antenna of the palp
16
50

16

14
40

14

12
12
30

10
10
20

8
8
10

D. aciculata D. neapolitana D. aciculata D. neapolitana D. aciculata D. neapolitana

Length of the dorsal cirrus Ratio of the lengths of the dorsal cirrus Ratio of the lengths of the dorsal cirrus
at the 10th chaetiger in mm and the branchiae at the 10th chaetiger and the parapodium at the 10th chaetiger
0.3 0.4 0.5 0.6 0.7 0.8 0.9

5.0
4.5

4.0
3.5

3.0
2.5

2.0
1.5

D. aciculata D. neapolitana D. aciculata D. neapolitana D. aciculata D. neapolitana

F I G U R E 4   Box plots of the nine characters, which were highly significantly different between pre-assigned D. neapolitana and D. aciculata
specimens
 |
12       ELGETANY et al.

(a) (b)

4 ASA3 D. aciculata
D. neapolitana
Dimension 2 (12.44%)
2

10
NO4

8
MMD index
0

6
AType

4
–2

NPara2 Dvou D. aciculata D. neapolitana

interspecific intraspecific

–6 –4 –2 0 2 4 6
Dimension 1 (30.94%)
(c) (d)
4

ASA3
D. aciculata
ASA5

12
2

ASA2
Dimension 2 (12.78%)

10
NType

MMD index
8
NPara1 NO4
0

6
NPara3 AAMS40 AType

4
NPara2 AAMS10
CS2

2
AAMS2
−2

Dvou D. aciculata D. neapolitana

interspecific intraspecific
D. neapolitana NE2
AE1
−4

–6 –4 –2 0 2 4 6
Dimension 1 (23.92%)
(e)
6

(f)
ASA3
ASA5
D. aciculata
4

AAMS40
12

ASA2
Dimension 2 (13.35%)

NO4
2

NPara1
10
MMD index

NType
8
0

NPara3 AType
4

NPara2 Dvou AAMS10

–2

Legend
CS2 D. aciculata D. neapolitana
Australia interspecific intraspecific
–4

South Africa
D. neapolitana NE2
AAMS2
AE1
Egypt
Spain/Italy
–6

–6 –4 –2 0 2 4 6
Dimension 1 (24.77%)
ELGETANY et al.
F I G U R E 5   Plots of the principal component analyses and box plots of the MMD indices between and within pre-assigned D. neapolitana
and D. aciculata specimens. Different strategies to ameliorate missing data have been applied: (a, b) deleting all specimens with missing data, (c,
d) assuming average values for the missing values and (e, f) imputing the values of the missing data. Colours indicate species and regions. The
percentage after the dimensions provides the contribution of the dimension to the overall variability in the data
of each other. For example, three of the nine characters are
related to the size of the dorsal cirrus at the 10th chaeti-
ger. Hence, the nine characters could possibly be grouped
into four suites of potentially dependent characters: length
variation of the head appendages (i.e., antennae and palps),
length variation of the dorsal cirrus at the 10th chaetiger,
shape of frontal lips and first appearance of subacicular
hooks. In D. neapolitana, head appendages are generally
shorter with fewer rings than in D. aciculata, and the fron-
tal lip is short and thick instead of long and thin (Table 3).
Similarly, the dorsal cirrus at the 10th chaetiger is smaller
in D. neapolitana, thus supporting Rodrigues et al. (2009),
and the subacicular hooks appear later.
Dagli et  al.  (2005) mentioned some possible additional
differences between the two species. Firstly, the nuchal or-
gans are rounded in D. aciculata or sub-triangular in D. nea-
politana. Secondly, the peristomial cirri are about three times
longer than the peristomium in D. aciculata and of the same
length in D. neapolitana. Thirdly, the ventral lobe is present
on different chaetigers in the two species, and finally, max-
illae I and II are of slightly different shapes. However, they
did not statistically assess these differences with respect to
intraspecific variation. Given the limitations of our material,
we could not assess the shape of the nuchal organs and max-
illae, but we could compare the length of the peristomial cirri
and the presence of the ventral lobe (Table S2). Only the last
chaetiger bearing ventral lobes is significantly different be-
tween the two species.
Another character, which was recently proposed to sup-
port the validity of two species, is the presence of paired
spermaducal papillae in D. neapoliatana only (Arias
et al., 2016; Bergamo et al., 2019). Diopatra neapolitana
are protandric hermaphrodites and display this character
only during their simultaneous phase of hermaphroditism,
when the specimen transitions from the male phase to fe-
male one (Arias et al., 2016; Bergamo et al., 2019). So far,
this character is not known from any other Diopatra spe-
cies and might therefore support differentiation of these
two species. However, due to the state of preservation
of some specimens examined, we could not observe this
character unambiguously across all specimens, including
D. neapolitana, and consequently did not consider it in
this study. Another challenge is that this character is not
well observable in immature individuals, mature males or
mature females (Bergamo et al., 2019). Hence, absence of
this character is only taxonomically useful if it is certain
that the specimen examined is a simultaneous hermaphro-
dite. We could not always determine this. Nonetheless, this
  
   13
|
character should be explored in future studies concerning
D. neapolitana and D. aciculata, including studies on the
reproductive biology of the latter.
When specimens were pre-assigned to one of the two
species, differences between the species seem to be evi-
dent. However, when specimens were not pre-assigned,
these clear differences disappear and the principal com-
ponent analyses cannot clearly separate the two species.
In the hierarchical clustering analyses, specimens of one
species grouped together with specimens of the other. In
particular, in these analyses, the Egyptian specimen NE2
is morphologically more like D. aciculata than D. neapol-
itana, to which it is assigned based on the mitochondrial
markers. Investigating the nine characters that differ sig-
nificantly reveals that several specimens in the overlapping
area of the principal component analysis (Figure  5e) are
more similar to the species to which they were not pre-as-
signed (Table  3). For example, in the Egyptian specimen
NE2, the subacicular hooks first appear at 17th chaetiger,
which is much closer to the median of 16 of D. aciculata
than the 21 of D. neapolitana. Similarly, the lateral anten-
nae reach back to the 9th chaetiger, which is the median of
D. aciculata. The five specimens pre-assigned to D. acic-
ulata in this overlapping area all have dorsal cirri at the
10th chaetiger which are as small as or smaller than the
median for D. neapolitana. Hence, while the two species
can be statistically separated from each other using certain
morphological characters, individual specimens cannot be
unequivocally assigned to the supposed species. This be-
comes especially evident for the specimens from Egypt,
which occur in sympatry but are seemingly different spe-
cies based on mitochondrial data. However, their morpho-
logical assignments are also not certain as each specimen
shows characteristics of both species.
Unfortunately, the molecular data also did not allow for
a clear separation into two species. The maternally inher-
ited mitochondrial markers COI and 16S generally separate
the two species albeit with low support and not necessar-
ily as reciprocally monophyletic. This would be expected
given the phylogenetic species concept (Cracraft,  1989).
By contrast, the nuclear markers 28S, ITS1, and ITS2 do
not support such a separation. The 28S is identical across
all sampled D. aciculata and D. neapolitana specimens.
This is a slowly evolving gene, and it is possible that not
enough time has passed since the speciation event for ge-
netic differences to accumulate within this marker (De-
Queiroz,  2007; Hausdorf,  2011). Similarly, the Egyptian
specimens possess the same ITS2 sequence, even though
 |
14       ELGETANY et al.

(a) NPara2 CS3 (b)

NS1 NS1
CS3 NPara2
NO1 NO6
NO7 NO1
NO8 D. neapolitana NO7
NO6 NO8
NO3 NO2
NO4 NO4
NO5 NO3
NO2 NO5
AAMS26 ASA3
NS2 AAMS17
ASA3 AAMS37
AAMS3 AAMS18
AAMS5 AAMS22
AAMS4 Dvou
Dvou NS2
AAMS34 AAMS26
AAMS32 D. aciculata AAMS4
AAMS30 AAMS3
AAMS6 AAMS5
AType AAMS6
AWAM2
AAMS18 AAMS32
AAMS22 AAMS34
AAMS17 AType
AAMS37 AWAM2
5 4 3 2 1 0 2 4 6 8 10
3 2 1 0 0 2 4 6 8 10 12

CS1 NS2
CS3 NType
CS2 NS1
NType NS3
NS2 NO8
NS1 NO5
NS3 NO2
NO5 NO4
NO2 NPara2
NO8 D. neapolitana NPara3
NO7 NO3
NO1 NO6
NPara2 NO1
NO3 NO7
NO6 AWAM1
NPara3 AType
NO4 AWAM2
AAMS8 AWAM3
ASA2 AWAM4
ASA3 AAMS8
ASA5 ASA2
AAMS40 ASA3
AAMS11 ASA5
NPara1 AAMS15
AAMS25 AAMS25
AAMS15 NPara1
AAMS26 AAMS40
AAMS27 AAMS26
AAMS16 AAMS27
AAMS7 AAMS23
AAMS24 AAMS16
AAMS23 AAMS24
AAMS19 AAMS22
AAMS18 AAMS39
AAMS22 AAMS18
AAMS39 AAMS19
AAMS10 AAMS13
AAMS14
AAMS9 AAMS11
AWAM2 AAMS20
AWAM3 AAMS21
AWAM4 CS3
AAMS2 CS1
AAMS13
AAMS14 D. aciculata CS2
AType Dvou
AWAM1 AAMS9
AAMS17 AAMS10
AAMS35 NE2
AAMS41 AAMS2
AAMS37 AAMS3
AAMS29 AE1
AAMS36 AAMS38
AAMS28 AAMS34
AAMS31 AAMS32
AAMS20 AAMS33
AAMS21 AAMS6
Dvou AAMS30
NE2 AAMS5
AAMS1 AAMS12
AAMS34 AAMS17
AAMS32 AAMS35
AAMS33 AAMS41
AAMS4 AAMS7
AAMS28
(c) AAMS3 AAMS31 (d)
AAMS38 AAMS36
AE1 AAMS29
AAMS6 AAMS37
AAMS30 AAMS1
AAMS12 AAMS4
AAMS5
ELGETANY et al.
F I G U R E 6   Hierarchical clustering of the pairwise distances between the specimens in the principal component analyses based on the first
two components (a, c) and all components (b, d) explaining together 95% of the variability. (a), (b) All specimens with missing data deleted; (c),
(d) values of the missing data imputed. The depth of the distance between the specimens is specimens are indicated by the scale bars. Specimens
deviating from the pre-assignment in their position in the hierarchical clustering are indicated in bold and with arrows. Results of the strategy to
average missing values are shown in Figure S1
they are placed in different species using mitochondrial
markers. In contrast, in ITS1 the two Egyptian specimens
are quite different, but specimen AE1 is more similar to
D. neapolitana and NE2 to D. aciculata, which again con-
tradict their positions using mitochondrial markers. Hence,
the biparentally inherited nuclear data could either indicate
ongoing gene flow between these two species, or that the
speciation event was really recent and the conflict is due to
incomplete lineage sorting.
The results presented herein do not unequivocally
support the presence of two species, nor their syn-
onymization. Applying a morphological species concept
(Cronquist,  1978) could justify maintaining two species
as statistically significant differences can be detected in
four character suites. Similarly, results from the mitochon-
drial markers would allow separating two species accord-
ing to genetic (Baker & Bradley,  2006) and phylogenetic
(Cracraft, 1989) species concepts. However, strong differ-
ences between the two species mostly occur in allopatry,
while in the only case of sympatry, i.e., the two specimens
from Egypt, the differences are much less pronounced or
disappear in some of the molecular markers capable of re-
combination. Hence, observed differences between the two
species could also be due to strong population structure in
the presence of allopatry for both molecular and morpho-
logical data (Sukumaran & Knowles, 2017). Accordingly,
biological species concepts relying on reproductive isola-
tion in sympatry (Mayr,  1942; Styan et  al.,  2017) do not
necessarily support the presence of two species. However,
in the light of genomic data, renewed interest in, and debate
around, speciation processes in the presence of ongoing
gene flow has been ignited and species have been recog-
nized as separate relatively early in the speciation process
(Coetzee, Hunt, Wilkerson, Coulibaly, & Besansky, 2013;
Feder, Egan, & Nosil, 2012; Foote, 2018; Harvey, Singhal,
& Rabosky, 2019). In summary, our results indicate that the
speciation event separating D. neapolitana and D. aciculata
is probably young and the species are in the so-called grey
zone of speciation (De-Queiroz,  2007; Hausdorf,  2011).
Considering taxonomic stability and the uncertainty of
our results, we decided to not synonymize the two species
but keep them separate. However, it is obvious that more
data are needed to address this topic in more detail. These
should comprise more, faster evolving molecular markers
as well as additional specimens, especially from possible
contact zones such as the Suez Canal (Egypt). Especially
interesting in this respect would be samples from along the
  
|
   15
Eastern African, Indian and the South-Eastern Asian coast-
lines as these might be additional sympatric populations.
Another interesting aspect of both species is that they
show wide distributions with seemingly recent dispersal
across oceans. Diopatra aciculata is not only restricted
to Australia as previously assumed (New South Wales,
Victoria, South Australia, Western Australia; Paxton, 1993),
but also present in South Africa and Egypt. Furthermore, if
early reports of D. neapolitana in South Africa (Day, 1967;
MacNae,  1957) represent misidentifications of D. acic-
ulata, then these reports predate the description of this
species in Australia, casting doubt on its presumed native
range. Regardless of the native range, shared mitochondrial
haplotypes between these distant regions could indicate re-
cent dispersal of D. aciculata, possibly by human activity.
Shipping and aquaculture, two important vectors of disper-
sal for non-indigenous polychaete species (Çinar,  2013),
were both implicated in the movement of Diopatra spe-
cies (Woodin, Wethey, & Dubois,  2014; Zenetos, Gofas,
Verlaque, Çinar, & Garcia Raso,  2010). Furthermore,
movement of exotic annelids for the bait trade has led to
establishment of annelid species in their recipient regions
(Arias, Richter, Anadón, & Glasby,  2013). There are,
however, no reports of either D. aciculata or D. neapoli-
tana being transported in this manner (P. Hutchings, pers.
comm., but see Saito et  al.  (2014) regarding transloca-
tion of D. sugokai around East Asia), although live bait
worms are sometimes transported illegally by fishermen (P.
Hutchings, pers. comm.). Distribution of D. neapolitana
is certain along the European coastline, which includes
the Mediterranean Sea and Atlantic Iberian and French
coasts (Arias et al., 2016; Berke et al., 2010; Pires, Paxton,
Quintino, & Rodrigues,  2010), and the Brazilian Atlantic
coast (Bergamo et  al.,  2019). Additionally, our analyses
included one specimen from the Suez Canal (Egypt) and
a GenBank entry from India, both of which shared mito-
chondrial haplotypes with the specimens from European
waters. Hence, our results provide some credibility to pre-
vious reports of D. neapolitana outside European waters
(Fauvel, 1953; Parameswaran, 1973; Wehe & Fiege, 2002).
Finally, molecular and morphological evidence presented
herein shows that mitochondrial sequence information de-
posited in GenBank as being from D. dentata was misidenti-
fied and is indeed D. aciculata. Diopatra dentata differs from
the other two species in several morphological characters in-
cluding relatively short antennae with median dark band and
bulbous tips, pseudocompound hooks with long appendages,
 |
16      

T A B L E 3   Values of the morphological characters with significant differences between D. neapolitana and D. aciculata for specimens, which were uncertain in their placement given the principal
component analyses

D. aciculata D. neapolitana
Number in
manuscript Character Significance level Median AE1 DVou AAMS2 AAMS10 AAMS40 Median NE2 NPara3 CS2
7 Median antenna ceratophore p < .001 14 14 12 12 14 13 12 12 NA NA
11 Length of median antenna p < .01 10 NA 8 7 NA 5 7 8 NA NA
8 Lateral antenna ceratophore p < .001 15 17 15 15 16 15 14 14 NA 14
12 Length of lateral antennae p < .001 9 NA 9 10 NA 6 8 9 NA 10
9 Palp ceratophore p < .001 14 15 11 14 14 15 12 12 13 15
13 Length of palp p < .01 3 NA 4 3 NA 3 2 NA 2 3
21 Frontal lips p < .001* 2 NA 1 2 1 1 1 1 NA 1
17 Ventral lobe last chaetiger p < .01 24 NA 30 18 17 27 28 NA 28 24
3 Dorsal cirri length 10th p < .001 3.25 2 2 2.5 2.5 2.5 2.5 2 2 2,5
chaetiger
4 Dorsal cirri to parapodium p < .001* 3 2 2 2.5 3 2.5 2 NA 2 2,5
5 Dorsal cirri to branchiae p < .001* 0.75 0.66 0.5 0.6 0.6 0.7 0.3 NA 0.35 0,6
18 Subacicular hook first p < .001 16 15 21 16 21 16 21 17 21 44
chaetiger
*Except for outliers no overlap in the box plots; bold values: clear assignment to the median of the other species; italic values: tendency towards the median of the other species.
ELGETANY et al.
ELGETANY et al.
mandibles with a single distal incision, having a smaller
number of pectinate chaetae and limbate chaetae with less
pronounced proximal shelves.
ACKNOWLEDGEMENTS
This work was funded by a stipend of the Egyptian Government
to AHE. We also would like to thank Audun Schrøder-Nielsen
and Lisbeth Thorbek of the DNA laboratory at NHM for their
valuable support and Ann-Helen Rønning for help with ship-
ping all the samples back to the different museums. We thank
the Museo Nacional de Ciencias Naturales, the Smithsonian
National Museum of Natural History, the University Museum
of Bergen, Western Australian Museum, the Australian
Museum and the Museums Victoria for providing the speci-
mens from their collections. CAS, HvR and CAM were sup-
ported by a National Research Foundation grant under the
Foundational Biodiversity Information Programme. Sampling
in South Africa was based on the permit RES2017-27 issued
by the Department of Agriculture, Forestry and Fisheries to
CAS. This is NHM Evolutionary Genomics Lab contribution
No. #18.
ORCID
Asmaa H. Elgetany  https://orcid.
org/0000-0001-6071-1953
Conrad Matthee  https://orcid.org/0000-0002-6836-069X
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SUPPORTING INFORMATION
Additional supporting information may be found online in
the Supporting Information section.
How to cite this article: Elgetany AH, van Rensburg
H, Hektoen M, et al. Species delineation in the
speciation grey zone—The case of Diopatra
(Annelida, Onuphidae). Zool Scr. 2020;00:1–19.
https://doi.org/10.1111/zsc.12421