cytogenetic arguments in favour of a taxonomic revision of lepilemur septentrionalis

Reviewed Article

Folia Primatol 2001;72:308–315

Cytogenetic Arguments in Favour ofa Taxonomic Revision ofLepilemur septentrionalis

Yves Rumplera Berthine Ravaoarimananaa,b Marcel Hauwya

Stéphanie Wartera

aEA 1315 Primates: Variabilité et Evolution des Prosimiens à l’Homme,Institut d’Embryologie, Faculté de Médecine, Strasbourg, France;bDépartement de Paléontologie et d’Anthropologie Biologique,Université d’Antananarivo, Madagascar

Received: November 17, 2000Accepted after revision: September 3, 2001

Dr. Yves Rumpler, Université Louis-PasteurEA 1315 Primates: Variabilité et Evolution des Prosimiens àl’Homme, Institut d’Embryologie, Faculté de Médecine11, rue Humann, F–67085 Strasbourg Cedex (France)Tel. +33 03 88 35 87 77, Fax +33 03 88 24 20 05

ABCFax + 41 61 306 12 34E-Mail [email protected]

© 2002 S. Karger AG, Basel0015–5713/01/0726–0308$17.50/0

Accessible online at:www.karger.com/journals/fpr

Key WordsCytogenetics W Lepilemur septentrionalis W Lemur W Chromosomal evolution W

Taxonomy

AbstractCytogenetic investigations performed on 30 specimens of Lepilemur septen-

trionalis confirmed the existence of 4 karyotypes differing from each other by 1–2chromosomal rearrangements. These data, pooled with those obtained in earlierstudies, showed that out of 60 animals karyotyped only two kinds of hybrids weredetected, allowing us to characterise two chromosomally polymorphic popula-tions. No natural hybrids could be found between these two populations, whichcould thus be considered as two separate species. The possible role of the chromo-somal rearrangements in the process of reproductive isolation between these twopopulations is discussed.

Copyright © 2002 S. Karger AG, Basel

Introduction

Since it has long been realised that most species of eutherian mammals differ intheir karyotype, cytogenetic studies have often been used for systematic purposes. In thelemurs, where morpho-anatomical and biogeographical criteria have occasionally led toconflicting classifications (especially in the family Lepilemuridae), cytogenetic tech-niques have been employed fruitfully to resolve such conflicts.

Thus in the Lepilemuridae, while morphological differences are not consistentenough to distinguish between different taxa, cytogenetic considerations have permitted

Cytogenetics of Lepilemur septentrionalis Folia Primatol 2001;72:308–315 309

6 species to be distinguished, differing from each other in numerous, complex chromo-somal rearrangements [1, 2]. These taxonomic investigations are of considerable inter-est as systematics is a necessary preliminary to any conservation effort. This is particu-larly important for Lepilemur septentrionalis, as hunting and increasing deforestationhave made this one of the most threatened species in its genus. L. septentrionalis hasbeen reported to include 4 subspecies [3, 4], which differ from each other by a smallnumber of simple chromosomal rearrangements (1–3): L. s. ankaranensis (2N = 36a;LSA) and L. s. andrafiamenensis (2N = 38; LSN) L. s. septentrionalis (2N = 34; LSS),L. s. sahafarensis (2N = 36b; LSH). However, LSS and LSH, and LSA and LSN give riseto natural fertile hybrids leading some authors to consider that they must all belong to asingle interbreeding population of L. septentrionalis [5, 6].

If we consider the latter point of view to be correct, natural hybrids should alsooccur between the four cytogenetic types. In this paper, in order to test this hypothesis,we present data based on both a larger sample size from areas previously explored andon animals caught in newly investigated parts of the distribution of L. septentrionalis.These data, we argue, support a revision of the classification of L. septentrionalisaccording to which the 4 subspecies should be classified into 2 species.

Materials and Methods

AnimalsDuring three successive field studies (1997, 1998 and 1999), 30 new specimens were caught for

karyotyping by one of us (B.R.) in different forests within the area of distribution of L. septentrionalis(fig. 1). The animals were caught in traps made from wire netting and were anaesthetised using aninjection of 0.2 ml of ketamine solution (Parke-Davis). A small ear biopsy was performed on a clean-shaven part of the ear, previously disinfected with alcohol. The samples were put in a small sterilisedtube containing a cryoprotective mixture of 10% DMSO in MEM culture medium, kept at ambienttemperature for 30 min and then stored in liquid nitrogen. After recovery from anaesthesia, the ani-mals were released at the point of capture. The capture sites are shown in figure 1 and the number ofspecimens captured at each site is reported in table 1.

KaryotypesFibroblast cultures were established from the skin biopsies and metaphase chromosomes were

obtained by standard techniques. Conventionally stained and R- and C-banded metaphases were pre-pared and analysed.

Results

As no animal could be found in the forest of Sahafary, which has been almostcompletely destroyed by charcoal-burning and the Lepilemur population destroyed byhunting, animals were trapped north of the forest of Sahafary, in the forest of Andra-hona and to the south, in the forests of Ankarongana and the forests of the Analameramountain chain. In the Andrahona and Ankarongana forests, karyotypes were identicalto those described earlier for Sahafary. Among the 11 animals karyotyped, we found 2karyotypes with 34 chromosomes, of which 3 pairs were submetacentric autosomes(LSS), 4 karyotypes with 36 chromosomes of which 2 pairs were submetacentric auto-somes (LSH) and 5 karyotypes with 35 chromosomes resulting from LSS ! LSH crosses(fig. 2a).

310 Folia Primatol 2001;72:308–315 Rumpler/Ravaoarimanana/Hauwy/Warter

Fig. 1. Distribution area of L. septentrionalis. The animals inhabit the forests north of the Ambilo-Andapa road.

Sahafary


Fig. 2. R-banded metacentric chromosomes of the hybrids between LSN and LSA (a) and of thehybrids between LSS and LSH (b). LSN possesses only 1 pair of metacentric chromosomes of smallsize, while LSA possesses the same pair, in addition to a large pair of submetacentric chromosomes.LSH possesses 2 pairs of metacentric chromosomes, while LSS possesses the same ones and, in addi-tion, the same large pair of submetacentric chromosomes found in LSA.

Table 1. Number of animals, their diploid chromosome number and the sites of capture

Location Year ofcapture

LSS2N = 34

LSH2N = 36

LSH!LSS2N = 35

LSA2N = 36

LSN2N = 38

LSA!LSN2N = 37

1972/73 4 3 71985 1 1 1

Ankarongana 1999 3 1 4Andrahona 1999 1Madirobe 1999 2

Montagne d’AmbreJoffreville 1971 2 1Andasibe Antsalaka 1997 1

AnkaranaFirst clift 1968 1Matsaborimanga 1968 1Matsaborimanga 1998 1Andrafiabe 1968 1Antanamisondrotra 1969 1 1 1Marovato 1998 1

AndrafiamenaBetsiaka 1985 1Andavakoerana 1985 1Ambilomagodro 1998 1 1Anjakely 1972 2Anjakely-Antsahabe 1998 1 1

Analamera 1998 11 1

Total 8 8 12 21 5 6


Table 2. Correspondences between the submetacentric chromosomes and their acrocentric homolo-gues of the 4 karyotypes of L. septentrionalis (LSE) and the presumed ancestor LSE

LSE LSN LSA LSH LSS2N=38 2N=38 2N=36 2N=36 2N=34

1 submetacentric 1 submetacentric 2 submetacentrics 2 submetacentrics 3 submetacentrics

1 1 2 2 316 16 1p 16 1p7 7 1q 8 1q

17 17 16 1p 2p13 13 13 1q 2q

In this as in earlier studies, the animals in the forest of Analamera had the samekaryotypes as those observed in the forests of Ankarana, Montagne d’Ambre andAndrafiamena. Among the 19 animals studied, we found 16 karyotypes with 36 chro-mosomes (LSA) of which 2 pairs were submetacentric autosomes, 1 karyotype with 38chromosomes (LSN) of which 1 pair was submetacentric autosomes and 2 karyotypeswith 37 chromosomes, which were natural hybrids of LSN ! LSA crosses (fig. 2b).

These results are pooled with those of the earlier studies in table 1. The correspon-dences between the different chromosomes are shown in table 2. One submetacentricchromosome is present in the karyotype of the 4 subspecies (LSN 1 = LSS 3 = LSA 2 =LSH 2), while a natural polymorphism involves the metacentric chromosomes LSA 1and LSS 1 and their corresponding acrocentric chromosomes LSH 8 and LSH 16, aswell as LSN 7 and LSN 16, respectively.

Discussion

That L. septentrionalis is one of the most threatened species of Lepilemur is drama-tically confirmed by the impossibility of finding a single animal in the remaining part ofthe forest of Sahafary, where both LSS and LSH were discovered 20 years ago. Sincereports have differed concerning the existence of subspecies, further taxonomic investi-gations are required for this species as an essential preliminary to any conservationaction. The data reported in table 1 show that of 60 animals studied (30 in this studyand 30 in earlier ones), 13 natural hybrids were found between LSS and LSH and 6between LSN and LSA. No other heterozygote could be detected. The animals of Saha-fary, Ankarongana and Andrahona, LSS (2N = 34) and LSH (2N = 36), seem to consti-tute an interbreeding population with numerous hybrids 2N = 35. Similarly, the ani-mals of the forests of Montagne d’Ambre, Ankarana, Andrafiamena and Analamera,LSA (2N = 36) and LSN (2N = 38), seem to belong to another interbreeding populationeven though more animals with a diploid number 2N = 36 were observed in the Anala-mera forest, while the 2N = 37 hybrids were less numerous.

The absence of hybrids between the group with 34, 35 or 36 chromosomes, inwhich all animals contain a small metacentric chromosome acting as a marker, and thegroup with 36, 37 or 38 chromosomes suggests the existence of a reproductive barrierpreventing cross-fertilization and provides strong arguments in favour of a division into


Fig. 3. R-banded karyotype of LSN, which can also be considered as the ancestral karyotype of L. sep-tentrionalis.

2 species, L. septentrionalis and L. andrafiamenensis, both of which are characterised byan intraspecific chromosomal polymorphism.

The chromosomal evolution at the origin of this speciation could have occurred intwo different ways, either by Robertsonian translocations or by centric fusions. Accord-ing to the first possibility, the ancestral LSE (L. septentrionalis) karyotype would havebeen similar to that of LSN, containing 2N = 38 chromosomes, among them a submeta-centric LSE 1 (fig. 3). A Robertsonian translocation would have occurred between chro-mosomes LSE 7 and LSE 16 giving rise to an intraspecific chromosome polymorphismin the whole population. A second Robertsonian translocation, between chromosomesLSE 13 and LSE 17, would then have occurred in the north-eastern part of the distribu-tion area involving the Sahafary and Ankarongana forests. It would then have becomehomozygous, marking separation between the 2 species (fig. 4).

According to the second theory, the ancestral karyotype of LSE would have beensimilar to LSS, containing 2N = 34 chromosomes, of which 3 pairs were submetacentricchromosomes. A centric fission would have occurred on LSE 1, giving rise to 2 acrocent-ric chromosomes. This rearrangement would have generated an intraspecific polymor-phism, and a second centromeric fission would then have occurred, spreading in the


Fig. 4. Schema showing how a polymorphic population could have been separated into 2 species, eachremaining polymorphic.

south-western part of the distribution and becoming homozygous in the areas of Mon-tagne d’Ambre, Ankarana, Analamera and Andrafiamena.

Whichever occurred, the second karyotypic rearrangement could thus be consid-ered either as a marker of the reproductive barrier existing between the two species or asa cause of speciation. According to the first hypothesis, speciation could thus haveoccurred in two different ways: (1) the fertility of the hybrids could have been reduced asa result of the karyotype heterozygosity, hybrid unfitness would then have promotedgene changes leading to speciation; (2) karyotypic differences could have acted so thathybrids were sterile purely as a result of meiotic disturbance so that any genic differ-ences between the two populations would not have had to contribute to sterility.

Robertsonian heterozygotes with a long meiotic chain configuration have oftenbeen reported as a cause of reduced hybrid fertility [7, 8], especially in lemurs [9, 10].However, chromosomal speciation can also occur, even in the case of a unique chromo-somal rearrangement, because gene differences between the populations could accumu-late in response to even a small degree of unfitness, the same rearrangement acting in adifferent manner depending on the genetic background of the karyotypic heterozygote,as has been well studied for the Rob t(13;21) in human pathology [8, 11]. It is moreplausible that karyotypic differences could promote speciation simultaneously with gen-ic factors, as has been shown in both lemurs [9, 10] and mice [12], but we have also to


keep in mind that genic hybridism may also contribute to a reduction in fertility in amore independent fashion. The unique chromosomal rearrangement distinguishingboth species would then be considered more as a marker of the reproductive barrierthan a cause.

Conclusion

In conclusion, new cytogenetic investigations of L. septentrionalis confirm the exis-tence of natural hybrids within only 2 populations, which thus appear chromosomallypolymorphic. No natural hybrid could be found between these 2 populations and theymay, thus, be considered as 2 separate species. Taking into account these results, insteadof a single species, L. septentrionalis, comprising 4 subspecies, we should consider thatthere are probably 2 species, L. septentrionalis and L. andrafiamenensis, each character-ised by an intraspecific chromosomal polymorphism. Mitochondrial DNA studies inprogress should provide further evidence.

Acknowledgments

We thank Mrs. Marguerite Lavaux for secretarial assistance and drawing and Robin Cromptonfor correcting the English. This work was partially supported by the Association Européenne pourl’Etude et la Conservation des Lémuriens. We acknowledge the Commission Tripartite of the Malaga-sy Government and the Ministère pour la Production Animale et des Eaux et Forêts for their permis-sion to capture the animals and to take samples.

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cytogenetic arguments in favour of a taxonomic revision of lepilemur septentrionalis

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