= CHROMOSOMAL SPECIATION IN A PROECHIMYS, RODENTIA, ECHIMYIDAE) eR Onvaldo A. REIG!, Marisol AGUILERA, Maria Alicia BARROS', and Myrian/USECHE? ode * Depart ‘udios Ambientales, Universidad Simé: Bolivar Aptdo. 80.659, Caracas 108, Venezuela * Departamento de Biologia, Facultad de Ciencias, Universidad de Los Andes, Mérida, Venezuela DDA series of populations of spiny rats (Procchimys) of NW and N Central Venezuela were found to belong to A circle of species and subspecies which surrounes the Andes of Mérida-Cordillera de la Costa mountain Nis. This ‘Rassonkre? consists of 6 succesive karyo, ‘morphs which exhibit a stepped eline distribution Ga = 42, 44, 46, 48, 50 and 62). Lach ofthe karyo- mocphs occupies'a diseeete subarea in an alnos co ‘fiiuous territory. Polymorphism is: very rare and no intermediate karyotypes have been found in the bos. #8 ofeach subarea, Although contact zones have not been studied in detail, the distribution scems to be mostly parapatric, Areas ar, cases without effective the stops from 2n = 44) ym In = 42 t0 Qn = 44 Ia change and 3 pericentric inversions Siig from 2n = 50 to 2n = 62 requires’ 6 RoboTeOM fearrangements and 2 pericentsie inversions, It is in- ferred that successive populations of the 2n = 44 — 2n = $0 sores may be at an incomplete stage of repro. Guctive isolation by partial reproductive failure of the F, hybrids, Reproductive isolation may be almost complete between the 2n = 42 and 2n = 44 and the 98 and 2n = 62 karyomosphs. This ‘Rasseakreis '8 classified as a superspecies, Procehinys (guairae], comprising three allospecies: & poliopus, P. guairae, and 4 new unnamed species (Barina’s sp. n). Within iairae 4 subspecies (or semiapecies) ave recog. tized, 2of which are named (P. guairae guainae and P. fuciiae ochraceus). It is postulated that differentia u this superspecies occurred during the Late eistocere. The tentative interpretation is advanced that rapid colonization occurred over most of the pre * fertory by a chromosomicaly uniform invacing Genetica Vols. 5 stock, which subsequently became differentiated by Parapatric chromosome raciation. This was followed by further chromosome differentiation in peripheral isolates which formed in unfavorable, dry climatic Phases. During humid climatic phases the ranges ex. Panded and zones of secondary contact -with the ‘original populations were established. Introduction {t is now generally agreed among evolutionary biolo- sists that in many eases chromosome rearrangements Play 2 causative role in the speciation of sexually re- Producing organisms.’ ‘The theory of speciation based on classical popula- tion genetics, denied or underestimated the roleplay. ed by evtogenetic processes in the differentiation of speci, It has been the merit of Michael White (1978) > {o have presented the many cases in which changes at the karyotypic level leat to reproductive isolation. Nevertheless the theory of species formation by chromosomal reairangement is far from being settled. One question is, how much reproductive isolation can‘ ! be inferred from the various types of chromosome rear. Tangements? Other questions are’concerned with the se0graphie pattern within which chromosomal changes established. Do’all cases Of" ehromosomal speciation imply conditions of parapatry or sympatry, as postu {ated by the stasipatric model, or may karyotype rear rangements also cause reproductive isolation in popu lations which have been previously geographically iso. lated? Are allopatric species originated by chremo. some changes distributed as a result of invasion of new areas after their differentiation, or do their pre- 291sent areas correspond 1 the sreas of their original ilferentiation? ‘These, and similar questions cannot be uacquivo- ceally answered at present, and to do so will require ‘the analysis of new relovant exzmples. It is therefore of considerable interost to analyze a fairly well docu- mented example of yeographic variation in chromo- some numbe morphology which occurs among, populations of the forest-cwoiling spiny rats (Procchi- mys) of Venezuela. As this example shows different degrees of clinal chromesomal differentiation within is of closely related forms, it ssems partic- ove questions. The ease presont authors a Rasson ularly reievast to tackle the sidy boon advanced by th (Reig ef al., 1978), but it is here presented in-detail Bin and extensively discussed forthe frst time. lection emoly served 1 Material and methods ages We studied 163 specimens of Procchimys, sampled in. (Ne Ut 57 different toca of NW and N Cent Venenucla | eel (Table 1, Fig. 1). Most of the specimens were pro- as cessed for chromosome analysis in the laboratory at ie the Simén Bolivar University. A few were processed = | fruit atthe Cytopeetts Larson of the Deparment ot REwato Biology, University of Los Andes in Mérida. After en, tissuesampling, te animals were prepared as skin end rye sf after me; " i ’ fa “ from eae) CARIBBEAN SEA init mosome, similar CS ° bi dee means off a fences vec: pooling th ulations. ss) Tuate chr types. Di nificant i ‘shared’ (6) jv + somes or means of S| and differs ions were a + Salon Fos the « the autosom to the posit Sizes. Setacer ‘elocentrie el Levan, Frede and small cate to > 7%, bent of the total ler “Group A eon their controaie A toestities of Karyotye: sized to small cy sigs correspond to localities named in Table 1 ° 300 200 ; EB + somes: group C = ~ “ a 5 telocentrie anc Vonsnisit, showing the distrivution of karyonoephs oF ths Rassenkreis of the Proeehinys — sonosomes mal mental number arms of the auic| ‘ i skull voucher specimens, and are deposited in the col- Jection of Mammals of the above institutions. Serum, hemolysates and samples of other tisues were pre served for parallel electrophoretic studies. Chromosome preparations were performed using the current bone-marrow technique. Testes of misle specimens were processed using the procedure pres- eribed by Ford & Evarts (1969), and several speci- mens were also processed following the corneal epi thelium technique (Fredga, 1964). Metaphase con- figurations were photographed with a M-20 Wild automatic photomicroscope, and karyotypes were constructed from enlarged prints. Idiograms of each of the six recognized karyomorphs were calculated after measuring a minimum of 10 selected karyotypes from each locality. The relative lengths of each cheo- mosome pair are expressed as a percentage of the length of the haploid autosomal set plus one X chro: mosome. Idiograms of different localities showing similar karyomorphs were statistically compared. by ‘means of Student's test. When no significant differ jogram was constructed the two pop- ences oscurred, a single pooling the numerical inforntation fro uldtions. These idiograms were then compared to eva- uate chromosomal changes in the different karyo- types. Direct comparisons and pairwise tests of sig- nificant differences in mean values of supposed ‘shared (Bianchi & Bianchi, 1968) whole chromo- somes or chromosome segments were made again by means of Student’s # test. Sécondary constrictions and differentiated heterochromatic chromosome re sions were also used to supplement the karyometrical evaluation. For the description of the karyotypes, we sorted the autosomes into morphological classes according to the position of the centromere and to theit relative sizes. Metacentric, submetacentric, subtelocentric and telocentric chromosomes were defined according to Levan, Fredga & Sandberg (1964). Large, medium, and small categories were also defined and correspond to > 7%, between 4 and 7%, and <4%, respectively of the total length of the haploid female complement Group A comprises only large atitosomes, whatever their centromeric index; group B comprises medium: sized 10 small metacentrie and submetacentric auto- somes; group C comprises medium-sized to small sub- telocentric and telocentric autosomes. Finally, the gonosomes make group by themselves, The Funds ‘mental number (FN) is expressed as the number of arms of the autosomal set. Results Description of the karvotypes Six cifferent karyotypes, and a few minor variants in some of them, were found among the 37 sampled Populations. They range from 2n = 42 to 2n = 62 chromosomes, with Tour intermediate types of 2n = 44, 2n = 46, 2n = 48 and 2n= $0. Karyotype I. 2n= 42, FN = 76 (Fig. 2: Fig. 9-1) Group A. = 4 pies of autosomes: One pair af larsé metacen trict (A,) comprising 1.642 of the haploid genome, and 3 pais of submetscentries (Ay-A,) of dvervasing site (9.04%, 8.29% and 6.58%), Pais A, has 2 hewropyenotic band in the nile of the tong sant that is wisible im most preparations. Group Ih =U pairs of medivaysized ta small metacentsies snd suivmeotacentriex that gradually decrease in size Crom 6.22% to 1.88%, A secondary eunstretion aceurs on the Ton ste of the fourth pal Group C. —S pairs of untosomes: 3 pairs of sm i to 3.26% irs of 1 ub ‘and 2 tues that vary in size Foor 3.7 smnall telocentries X. = A mediunt sized (5,325) subtelocentrie, Vs = A small sized (1.45%) teloconttc. Karyotype Il. 2n = 44, FN = 72 (Fig. 3: Fig. up A. = 3 pire of auiosomes: I pair of large (Act: 11.77%) metacentsies, and 2 pace of large (A-2: 8.95%: A 8.16%) subleloceairics. There is @ heteronyenotic rexion fn the middle ofthe long sei of pits A-2 and Ae that i visible in most preparations Group B.~ 10 pairs of medium-sized to small motacentris and submetacentries ranging in size from 6.35% (Bel) to 2.32% (B10), The fourth pair has a secondaty constriction ‘othe long arn, a fe a an 0 RR Ba Pik §) clo) en oxH eet eee |» Ur a Tis. 2. EN 2700 marrow prepsta karyotype of karyomorp 1 chins pines), sme, Zl ns Giemsa sainA Gg ah gS my ee we c BE sn aa sa, oo ere an satstive aryoure of aryomerph IE Qn ) (Hrovehinys guairee acizaceus). Caja Seo. masroW pteparatioa; Giemsa stain. ma Se 2° Gs e ho 2a ao be xy Fig. 4, Representative karyotype of Karyemorph IIL @n = 45, FX = 72) of individual showing dition of the gonosumes, (roscitinse guairoe guairae). La one -narrow pisparation, Giemsa stain S pairs of autosomee: 1 mediumsized (Ct small (C4 3.7%) subtolocentrics, and 6 telo- in sie from mediamsized (C2: 4.17%) to =) Group ¢. 6.51%), and ni sined (3.9 saracierizes the population from El ic varistion was found in the size 19%) in specimens from nos ) tocentrie This karyotype 72.(Fig, 4; Fig. 9-111) tong A.=2 pits of autasomes: Ipair of large (AL 11.6.2) metacentrice and 1 pair of large (A-3: 8.52) sub Karyatype Ill. 2n = 46, FN Gaoup B= 10 sits of metiunesize And subietacentrics zonging in size’from 6.39% 2.27% (U-10), Tae fourth pair bears a secondary constriction Group C= VO pie of autosomes: | palt of medium-sized (C1: 6.63) telocentees that has a neat heteropycnatic region (ig. 8), Upsir of mediunesced (C2: 6.32%) and 1 pair of small (C5: 3.41%) sublelocontrics and 7 pairs of telocentrics fom 4.31% (C3) to 1.16% (C10). subtelocenttic. Ku» A medium sted (6.62 = Anal (97% tlocentic This ciiromosomal pattern was consisiently found, for the avtosomes, in all individuals of ie six sam- pled’ populations (Table 1). However, « polymer- phism for a ¥ chromosome isomorphic with the X ‘chromosome was found in the two males from Bahia do Cata (Reig, Kiblisky & LObig, 1970) and in 2 out of the $ males studied in the sample of La Trilla. A normal karyotype with a heteromorphic Y clromo- some was described by George & Weir (1975) from 5 males of a colony maintained at the Wellcome Insti tute of Comparative Physiology in London. This colo- ry was developed from specimens caught by the se- ior author of this paper in La Trilla. However, Goorge & Weir describe the ¥ chromosome as being i ng a ii a hagid Wotanahects Sakae zz onat itn rsd al ied eee aeT aa ae oe ae as me. tas aoe ied os sentative Karyotype of karyomorph IV (2n = 73) (roechinys guvirae, Miranda's subsp.). San iaanda Slule, Bone marrow preparse 48, FN Antonio (Lt Guapo), § ‘ion, Gienist sai.it) ‘Table 1 (wn per Pr neat ate reraet aera ster mine indintete wontnot et paties eras Sumterot E EE oie gies ae Sees ca fires: D Kismera, Zata Sete é ja 4276 eames T Erno, Zt State. ‘ Hi epnde EPS Nea candy Ctenbe . i ? I i 5 sarin deconbncTuchie Sate i 5 ie ase 1 | GQ nejuquero (Zea) Mévid State f 2 3a Ps AD Caja Seca, Mérida State L 2 44 re x Se aebarindste he teeeees f the X EICor . Lustia State, 2 44 > 1 Peneeama, Zulia State ss + Bobare, Lara State ' See ep snare Faleon State ec i San Esteban (Puerto Catto), Carabote State, PRR I ioor eee p from | 9 Ih ‘BS Bala de Cat, Aragua State- ii Rene uae Haat ga 1 Insti- Gcumare del Costs, Aragua Stat ata ategee an wis colo- f5SpLa Til, Aragia State Sim toa iss 7 Sees | (2) H1 Limén, aragea State. Gel ee nee fowerer, | GRY Nanvare, Carabobo State LOAM Gas Fae a being = 3) La Horqueta (Tat), Miranda Stat. mit he anh vie | TT BB Seen ren te an Cana ke 4 | Pisin cen be uvaneitesenl as THe Cees ete za oe Dos Camino, Guareo State ee haba ici go" ge ' (DY E10, Cojeds State. te meee ze hi Gmc eie ase t Big taal oh va ets + : ' Churcote, Cojedes State: eine eae | Hacienda’ La Coromoto (Apartaderos), Cojedes Sid sso 72 kL ELBaul, Cojedes State. eat i) (brn? ie | Nueva Florida, Portuguesa State, eo imiascg ta { Tur, Portuguese Sta Je Pens i San Trg, Porta State. vitae ey Selsip ta ja apewtere ee i + GB) Lastatas, Poroguets state. : 713) in en Peat 4) Las Cocutas,Portaguess State atone Mlsew ee I : S14 GB Rio Tecupido, Fortagrem Stat nen eaatiied iy t Ni As, G@ patns, nsrinas state, 21 4 Ge A TRinedn, Marina Stat hen ahora Buena Vist, Barinas State, ee ecd [hao Geng (Es Leng), Barn Sit, fins Agere Dy Guasialito, Apure State ctor x Si es AV Qn « a aE svsp). San GRAND TOTAL 9370163 rent 295ie, which is not in agreement with our re- nd with the general occurrence of telocentric ¥ ysontes in all the related chromosome forms ed in this paper. Disagreement in the auntber of telocentric and subtelocentric autosomes in former reports (Reig, Kiblisky & Labig, 1970, George & Weir, 1973} demonstrates the problem of interpreting he feaiures ef very small chrome: sates, Our present report settles the question after careful examination of moze than 100 selected metaphases. Karvotyge TV, 2n= 48, EN = 72 Pir Group Av ~ 2 pairs of astosorses: | pale wecentrics and I pair of laps (AS: 4 Fig. 91V) (1: 12.08) sovmetacen- Gros 8. =,9 pats of mediumeized 10 sm and submetaecntsies ranging i ste ftom, 6.225 (bl) 0 2.10% (Ib-9), ‘The fourth pair beats a secon. :¥ constriction, And although itis shorier than Ui third, itis placed in the ied order and named Bo a account ofits likely homolog with the fourth of kaeyotynes HIN, as discuss in section 4a Group C.— 12 pairs of autosomes: I pair of medium-sized (C-1: 6.89%) tslocenirics with 2 nes: Reteropyenotie regions pais of medium-sized (C2: 6.33%) and 1 pair of small (C5: 3.50%) subieloceniries; and 9 pairs of telocenties ranging From 4.188% (C3) t0 1.10% (C-12) X.— A medivmaized (6.18%) subtelocentic. Y. = A small 3.31%) telocentic All samples from San Antonio, Dos Caminos and El Limdn have this karyotype. A variation was found in specimens from Manuare, #1 P2o and Tierra Caliente This Karyotype of 2n =. 48, FN = 74 has 1 additional metacentrie in Group B and only 11 pairsin Group C. These differences can be accounted for by ane peri- cenizic inversion. The Y chromosome is also smaller (1.41%) inthe sample from Manuare. We shall call this karyotype IV-A. Karyotpe V.2n = 50, FN = 72 (Fig, 6; Fig. 9-V). Group A.—2 puite of sutosomes: 1 par of large (Ach 11.46%) melseenteics, and 1 pais of large (AI: 815%) sub- Group B. = 8 pairs off mediumsized t9 small metacentries fine submetacentees ranging in size ftom 5.15% (B-2) to 2.23 (B-11), The thind pair hoe a socondary constriction, land although itis shocter than the second, iti lased in the second order and named B-4 on secount of its Hikely homolo- fy with the fourth of karyoiypss Hit, as discussed i section Group C14 paiss of avtosonies: L gale of mediunesized (G12 6.592) telocenities wit 3 nest h fF medinessized (C2: 0020) a (C6: 3.37) suizviocentries; 2 96 eT meer a as Ty ay oKe ke c 88 G4 A. 2a 2 na pa 4a va nn xy Tig. 6. Representative karyotype of karyomorph V (2n = 50, FN = 72) Procchiny’sguairee, Lano's subsp). San Jorge, Portuguesa State, Hone marrow preparation, Giemsa stain. adhe ott BOX 40 wn 4s - cds 98 on so 4 ne AN oH kw tw rh ot. Kx xy rig. Te Representative karyotype of karyomorph V1 (20 = 62, FN = 74) (racchinys, Barina's new apvcies), Baiaitas arin State, Bone-marrow preparation, Giemsa stan Ceo, In \ hum and in t lifez ly fo and ¢ the § caugh comit (Hane 1000 chin Th sampl Caribt smerid into t tinuou Mérids of Nir “Cordil inits © Popome, ene i umsized (C3: 4.21%) and mediumsall (C4: 3.852) telo- cceniris; 5 paits of rather ltge small-sized telocentries alnvost indistinguishable in size (CS, C-7 to C10), and 4 pairs of Smittsized telocentries evenly decreasing in size (C-IL to C14), X.— Amudiumsized (5.81%) subtetocentee. Y.— Avery small (1.47) telacentrie, Karyotype VI. 2n = 62, FN = 74 (Fig. 7; Fig, 9-V1) Group A. 2 pairs of sutoromes: I pale of Lange (Al: 1241) metacentries, and | pair of large (AB: 8.87%) sub- Group B. —3 paits of small autosomes: 1 metacentric (D-10: 2.83%) and 2submetacentris (BAI: 1.99%); B-12: 1.70%) Group C. ~ 25 paits of autozomict: I pair of mediunvsized (CA: 6.04) telacentris with a neat heteropyenotic tezion; 1 reat micdiumaized subtelocentsic (C-2: $,$6%); 3 medium- Szod telocentrics showing small size decrease (C-3: 4.07 C4: 3.91%: C5: 3.8074) I pale of small subtetocentrics (C6 3.54%) and 19 pairs of small tolocentries grading evenly in size from 3.16% (C7) to 1.25% (C25). 3. — A medium-sized (5.70) metacentri, Y.= A very small (1.95%) telocentri. Geography and distribution pattern In Venezuela, spiny rats inhabit low-lend tropical humid forests, premontane and lower montane humid and very humid forests, and, occasionally, they occur in tropical dry forests (for our use of Venezuelan life-zones, see Ewel & Madriz, 1968). They are usual- ly found in moist sites in evergreen forests, but they are not infrequent near streams in brushy openings and eroplands. Spiny rats prefer lower elevation com- munities. In a sample of 459 individuals collected by the Smithsonian-Venezuelan Expedition, 75% were caught below 500 m and 96% below 1000 m, only 476 coming from localities between. 1000 and 1340.m (Handley, 1976:57).. Therefore, altitudes above 1000 m and xerophilous habitats are avoided by Proc- chimys and would act as effective dispersal barriers. ‘The populations reported in this paper have been sampled in NW and N Central Venezuela, between the Caribbean coast and parallel 7° 30'N, and between meridians 66° 10'W and 73°W. This area is divided into two major lowland regions by an almost con- tinuous mountain axis, This avis is formed by the Mérida Andes, the ‘serranias’ of Lara, the ‘serranias’ f Nirgua-Tinaquillo, and the Litoral Chain of the Cordillera de la Costa’ (Freile, 1971). The only break in its continuity is at the Yaracuy depression. Populations on éach side of this axis are separated by an almost continuous region higher than 500m above sea level. However, the altitudinal limit of 1000 m is interrupted by several pathways which con- nect the two sides of the mountain axis (Fig. 1). Pop ulational contacts may eccur through the Yaracuy depression and the lower mountain valleys of the ‘s¢r- ranias’ of Nirgus-Tinaquillo. Resides, continuity of the populations at each side of the mountain axis may oceur in the forested region between Higuerote and La Sebana, at the easternmost end of the Litoral Chain, There are no geographic barriers affeeting the continuity of the disteiUution of spiny rats along each side of the mountains. Therefore, the goographie dis- tribution of our samp'ed populations is that of an almost continuous belt that encircles a major moun- tain mass. The geography of the different parts of the belt is dealt with in the following description of the area of the different karyomorph. Populations having the same karyotype occur in discrete geographic areas. At present, these areas have been delimited with different dogrees of accuracy, but the pattem is clearly indicative of spatial group- ings of populations according to their karyotype (Fig. 1; Table 1). Each of these areas is characterized by an invariable, or slightly variable karyomorph. In- trapopulationa! polymorphism within in 2 given area, or interpopulational geographic variation in the same atea are nonexistent, with the few exceptions noted above. Some of these population groupings are allo patrie, but the majority appear to be parapatric. No cases were found where karyomorphs geographically overlapped, and we have not yet found intermediate heterokaryotypes in the contact areas. The successive chromosomally defined areas ex- hibit a step-cline pattern. Northwest of the eordillera diploid numbers increase from 42 (karyomorph 1) in the west to 48 (JV) in the cast; southeast of the thountains from 48 (IV) in the east to 62 (V1) in the ‘vest. From karyomorph I through V this inezease is by one pair of autosomes ata time; between the pa petrie karyomorphs V and VI there is an abrupt in cexease by six pairs (Fig. 1) Karyotype I occurs in the tropiea! humid forests of the lowlands and mountain slopes of W and SW Zulia State, in the basin of the Marzcatbo lake and from the eastern slopes of the Sierra de Perija to the slopes of westermost Mérida Andes, State We have only sampled populations fom twa locelities in this area (Fig. 1, Table 1). One (San Juan ee Co- 297