In: The Natural History of Madagascar (S. M. Goodman and J. Benstead, eds.) University of Chicago Press (in press)

Origin of Malagasy

Anne D. Yoder1,2 and John J. Flynn3

1Department of Cell and Molecular Biology Northwestern University Medical School Ward Building 303 E. Chicago Ave. Chicago, IL 60611

2Department of Zoology Field Museum of Natural History Roosevelt Road at Lake Shore Drive Chicago, IL 60605

3Department of Geology Field Museum of Natural History Roosevelt Road at Lake Shore Drive Chicago, IL 60605

Address correspondence to: Anne D. Yoder Department of Cell and Molecular Biology 303 E. Chicago Ave., Ward Building Chicago, IL 60611-3008 e-mail: [email protected] phone: 312-503-0005 fax: 312-503-7912 Introduction

As with the primates, the phylogenetic status of Malagasy Carnivora is of great significance for biogeographic theory generally, but most especially, for understanding the biogeographic history of Madagascar. Multiple phylogenetic origins, and thus multiple biogeographic invasions of Madagascar, have been proposed for the

Carnivora. This proposal is almost certainly due to the morphological and ecological diversity apparent among these with at least three distinguishable morphological and ecological types among the seven recognized genera of Malagasy

Carnivora. Four of the genera (Galidia, , Mungotictus, and ) are described as "-like" and their common names reflect this distinction (e.g.,

Galidia is known as the "Malagasy ring-tailed mongoose"). All four genera weigh between 600 - 900 g, have agile, low-slung bodies, short legs, and long bushy tails.

Crania are typically elongate and dorso-ventrally flattened with pointed muzzles; diet consists primarily of small vertebrates and insects (Albignac 1972; Albignac 1984) (see also pp. xx). The second morphological type, the Malagasy , comprises the two genera and . Although these two taxa are considerably different from each other, both are considerably larger than the mongoose types and have longer legs relative to body length. Eupleres is remarkable for its unusual diet that consists chiefly of earthworms (Albignac 1984) and for the concomitant suite of craniodental specializations. Fossa and Eupleres both demonstrate physiological specializations that allow for fat storage in the tail during Madagascar's winter months. The third morphological type, represented by the monotypic Cryptoprocta, is unique.

Commonly known as the "fosa" (not to be confused with the genus Fossa), Cryptoprocta weighs between 7-12 kg and is the largest member of Madagascar's extant Carnivora

(see pp. xx). It is a powerful predator and excellent climber, known even to hunt and

2 capture lemurs. Like , Cryptoprocta has retractile claws, but like , its gait is plantigrade (Albignac 1972).

Malagasy Carnivora diversity has been variously described by systematists.

Gregory and Hellman's (1939) extensive analysis of the morphology, evolution, and classification of "civets" placed the Malagasy Carnivora in four taxa: Euplerinae (close to

Asian hemigalines) and Fossinae within one lineage of , Galidictinae as a basal lineage of Viverridae, and Cryptoproctinae within . Simpson (1945) placed them all in the family Viverridae and defined seven genera within three subfamilies:

Galidiinae (the ), (including various Asian taxa with the

Malagasy civets placed in the monotypic tribes Fossini and Euplerini, and

Cryptoproctinae). Most recently, Wozencraft (1993) revised this arrangement by placing the Malagasy Hemigalinae into the Family Herpestidae (not recognized as a family by Simpson, 1945), raising the two monotypic tribes to subfamilies Euplerinae and Fossinae. Thus, at present, there are two families and four subfamilies that contain only seven genera. Furthermore, there is no consensus of opinion as to whether the various subfamilies are each more-closely related to continental Carnivora lineages

(thus implying multiple colonizations of Madagascar) or whether they comprise a monophyletic lineage (various opinions can be found in Pocock 1916; Gregory and

Hellman 1939; Albignac 1972; Hemmer 1978; Albignac 1984; Wozencraft 1989; Veron and Catzeflis 1993; Veron 1995). In particular, the position of Cryptoprocta has been controversial (Flower 1869; Pocock 1916; Gregory and Hellman 1939; Beaumont 1964;

Hemmer 1978; Wozencraft 1989; Veron and Catzeflis 1993; Veron 1995). Thus, in addition to biogeographic and evolutionary implications, phylogenetic resolution can have significant consequences for Carnivora systematics as a whole. If, for example, the

3 Malagasy taxa are discovered to comprise a clade, then Wozencraft's (1993) designation of either Herpestidae or Viverridae (or both) becomes paraphyletic, profoundly affecting our understanding of higher-level Carnivora phylogeny and evolution.

Cladistic and/or genetic investigations of Malagasy Carnivora phylogeny have been extremely limited. Early chromosome phenetic analyses concluded that the group is diphyletic or polyphyletic, representing two different invasions of Madagascar

(Couturier et al. 1986; Razafimahatratra 1988), whereas a more recent DNA sequencing study (Slattery and O'Brien 1995) found that Fossa and Galidia comprise a clade. Taxon sampling in the second study was so limited, however, that the results do not significantly on the question of Malagasy Carnivora monophyly (i.e., Fossa was the only

«viverrid» and Galidia the only «herpestid» included in the study). Other notable studies to have addressed the phylogeny of Malagasy Carnivora were focused on Cryptoprocta and included a DNA hybridization (Veron and Catzeflis 1993) and a cladistic analysis of morphological characters (Veron 1995). These two studies came to differing conclusions. The DNA hybridization study found that Cryptoprocta has affinities within the Herpestidae rather than the Viverridae (Fig. 1) whereas the morphological study grouped it with the Felidae, as did Gregory and Hellman (1939) (Fig. 2). The morphological study found the Malagasy mongooses to belong with other herpestids, although the two Malagasy genera were shown to occupy unresolved positions at the base of a combined herpestid/hyaenid clade. Flynn and Nedbal's (1998) cladistic analysis of nuclear transthyretin intron I sequence data strongly supported linking

Galidia, the only Malagasy carnivoran included in the study, as sister taxon to other herpestids. Several observations can be made from a comparison of these studies: 1) an apparent conflict exists between morphological and molecular resolutions of

4 Cryptoprocta's position in the Carnivora phylogeny; 2) the placement of Malagasy mongooses within the Herpestidae may be well-justified on morphological grounds; and 3) the karyotypic and morphological data indicate that there may have been between two to four invasions of Carnivora lineages to Madagascar.

To resolve these biogeographic and systematic issues, we have performed phylogenetic analysis on DNA sequences from three unlinked genetic markers: mitochondrial DNA (specifically, the cytochrome b and ND2 genes), the nuclear gene transthyretin (TR, intron I) and the nuclear interphotoreceptor retinoid-binding protein

(IRBP, exon I). These markers were chosen because they are unlinked and should therefore provide independent estimates of Malagasy Carnivora history and also because they represent both coding (cytochrome b, ND2, and IRBP) and non-coding

(TR) sequences, thereby providing both functional and neutral characters for phylogenetic analysis. Moreover, all four genes have been demonstrated to be informative for questions of both deep and recent mammalian interrelationships

(Stanhope et al. 1996; Yoder et al. 1996; Zardoya and Meyer 1996; Flynn and Nedbal

1998; Yoder and Irwin 1999; Flynn et al. 2000).

Materials and Methods

DNA was extracted from either fresh or museum tissues for all Malagasy taxa; outgroup sequences were either extracted de novo or taken from GenBank (see Yoder et al., (in prep.) for voucher and accession numbers). All cytochrome b and IRBP DNA sequences were generated at Northwestern University while those for TR and ND2 were generated at the Molecular Systematics Laboratory at the Field Museum of

Natural History. This separation of lab procedures and reagents further assures that the

5 phylogenies generated for each gene are independent. Extractions and sequencing followed methods detailed in Yoder et al. (1996) and Flynn and Nedbal (1998). PAUP*

4.0b2a (PPC) was employed for all phylogenetic analyses as well as for bootstrap tests of node support (Swofford 1998).

Results and Discussion

In Figure 3 we illustrate the results of the maximum parsimony analysis of the four genetic markers. For both of the mitochondrial data sets, a step matrix was imposed that weights transversions 10 times more heavily than transitions (i/v10 weighting). This follows the observation that transversions occur considerably more frequently than transitions in the evolution of mitochondrial DNA (Irwin et al. 1991;

Wakeley 1996; Yang and Yoder 1999). For both of the nuclear data sets, all characters were equally weighted.

A number of results are universal to the four maximum-parsimony gene trees.

Most importantly for the focus of this contribution, Malagasy Carnivora are consistently shown to form a clade, and always with strong statistical support (bootstrap values between 71% and 99%). Within this clade, the mongoose "types" (Galidia, Galidictis,

Mungotictis, and Salanoia, in the cytochrome b data set; the first three genera in the other three gene trees) are shown to form a clade, with Galidia consistently resolved as the sister lineage to the other genera. Our specimen sample is such that we have three

Galidia individuals, each from different areas of Madagascar, here described as south, central, and north. Invariably, the south and central specimens form a clade that excludes the north sample, although for IRBP, because the southern is not included, the north and central form a clade. The south and central samples represent

6 G. e. elegans and the north sample G. e. dambrensis. In numerous external and morphological characters, these forms are distinctly different from one another.

Another result that is universal to the four data sets is the finding that Cryptoprocta and

Fossa are basal within the Malagasy Carnivora clade, though with differing resolutions of their relative positions for each gene. The disagreement among genes, indicating uncertainty, is also apparent in the low bootstrap support for the differing placements of these two taxa. Despite the slightly differing placement of various taxa in the four gene trees, the results can be taken as robust evidence that Malagasy Carnivora are derived from a single common ancestor that colonized Madagascar only once, sometime in the past. The difficulty in resolving the positions of Cryptoprocta and Fossa suggests a rapid basal diversification of three lineages within Madagascar.

Our results run counter to previous hypotheses that suggest that a minimum of two colonizations is needed to explain extant Malagasy Carnivora diversity. As discussed in the introduction, these hypotheses depend on previous classifications of

Malagasy Carnivora that place them in at least two separate families (Wozencraft 1993),

Herpestidae (Galidia, Salanoia, Mungotictis, and Galidictis) and Viverridae (Fossa, Eupleres, and Cryptoprocta). Although our taxon sampling is not complete at the genus level for all genes, lacking Eupleres and Salanoia for all but the cytochrome b data set (see fig. 3 legend for explanation of differential sampling), our sample nonetheless includes representatives of both putative families and at least one representative of the three morphological types for all four genes. The results are clear: each of the morphological types that currently reside on Madagascar has evolved from a single ancestral morphological type, and furthermore, that morphological type was of a general herpestid form. This conclusion is easily drawn from the observation that the gene

7 trees are nearly unanimous in showing that the Malagasy Carnivora nest within a herpestid clade. The only exception to this result is seen in the ND2 tree wherein the two felids ( and ) also fall within the herpestid clade. Bootstrap support for this result is very low, however, casting additional doubt on its accuracy.

To alleviate the possibility of sampling error in the individual data sets, sequences for overlapping taxa for each gene were combined in a single data set. Parsimony analyses were conducted on this combined data set with all characters weighted equally.

The results of the combined analysis are congruent with those discussed above, but have the advantage of providing impressive statistical support for virtually every node in the phylogeny. For the combined-data analysis, the same resolution of the Malagasy mongooses described above is found, though with 100% bootstrap support for each clade. The entire Malagasy Carnivora clade is also resolved with 100% bootstrap support, though the relative positions of Cryptoprocta and Fossa remain unresolved.

Sister to the Malagasy group is a clade containing the comprehensive sample of Asian and African herpestids. That clade is also resolved with 100% bootstrap support; the sistergroup relationship between the two clades is supported by a 94% bootstrap value.

The genus Crocuta (a hyaenid) is the sister taxon to that clade with 95% bootstrap support. This clade, along with a viverrid clade and a felid clade, form a comprehensive feliform clade, supported by 100% bootstrap support, wherein the genus Nandinia is clearly shown to be the basal taxon, as in Flynn and Nedbal (1998), with 98% bootstrap support for its basal position. Our results therefore lend further evidence for the conclusion that the traditional "Viverridae" is a parapyletic taxon given that felids (Felis and Panthera, in our sample) and herpestids clearly nest within a putative Viverridae.

8 In summary, the results of this study show that the Malagasy Carnivora evolved from a single herpestid ancestor that probably emigrated from Africa (according to the geographic distribution of their herpestid relatives), sometime during the late Oligocene to early Miocene (30-20 mya). Thus, in comparison to various other groups of Malagasy vertebrates, the arrival of Carnivora to Madagascar has been relatively recent (Yoder et al. in prep.), though their rapid radiation into the present variety of morphological and ecological types is no less spectacular.

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13 Figure Legends

Figure 1

Tree based on DNA hybridization data (figure adapted from fig. 4 of Veron & Catzeflis

1993). Bold italics highlights Cryptoprocta.

Figure 2

Strict consensus of 27 equally parsimonious trees from analysis of 77 morphological characters (adapted from fig. 8 of Veron 1995). Malagasy Carnivora are indicated by bold italics.

Figure 3

Four gene trees determined by maximum parsimony analysis. Mitochondrial gene trees are on top row; nuclear gene trees are on bottom row. A) Cytochrome b gene tree wherein transversion were weighted 10 times more heavily than transitions. Note that cytochrome b tree includes Eupleres and Salanoia. These sequences were acquired from museum skin samples that were not analyzable for the other three genes due to poor DNA quality and quantity. B) ND2 gene tree wherein transversion were weighted 10 times more heavily than transitions. C) IRBP gene tree wherein all characters were equally weighted. D) Transthyretin gene tree wherein all characters were equally weighted. Bold italics highlight Malagasy taxa. Numbers represent bootstrap values. Circled bootstrap value indicates support for Malagasy Carnivora clade. Clades without illustrated bootstrap value received less than 50% bootstrap support.

14 Felis Felis catus Panthera Genetta genetta Genetta servalina Paguma Crocuta Cryptoprocta Ichneumia Canis Martes Nandinia Panthera Felis Cryptoprocta Genetta Proailurus Palaeoprionodon Hemigalus Paradoxurus Proionodon Civettictis Eupleres Fossa Salanoia Mungotictis Galidia Galidictis Suricata Cynictis Ichneumia Mungos Proteles Crocuta Tungurictis A) Cytochrome b (iv10) B) ND2 (iv10)

Homo Homo Cryptoprocta ferox Cryptoprocta ferox Fossa fossana 100 Galidia elegans (C) 82 Eupleres goudotii 100 Galidia elegans (S) 100 Galidia elegans (C) 100 Galidia elegans (N) 100 Galidia elegans (S) 71 94 Galidictis fasciata 100 Galidia elegans (N) Mungotictis decemlineata 60 98 Galidictis fasciata Fossa fossana 84 Salanoia concolor Mungotictis decemlineata 100 Felis silvestris 95 Crossarchus obscurus Panthera leo 72 Mungos mungo 55 Crocuta crocuta 100 Suricata suricatta Crossarchus obscurus 94 88 Cynictis pencillata Suricata suricatta 98 Herpestes edwardsii 100 Mungos mungo Herpestes javanicus 73 98 Cynictis pencillata 100 Felis silvestris Herpestes javanicus Panthera leo 100 Herpestes edwardsii 90 Nandinia binotata 98 Civettictis civetta 96 Civettictis civetta 85 58 Viverra tangalunga Viverra tangalunga 96 Genetta servalina 52 80 Hemigalus derbyanus Hemigalus derbyanus Paradoxurus 65 Genetta servalina Paradoxurus Crocuta crocuta Nandinia binotata Canis lupus 99 Tremarctos ornatus 63 Tremarctos ornatus 64 lotor Erignathus barbatus Erignathus barbatus Scalopus Canis lupus Procyon lotor Scalopus

C) IRBP D) Transthyretin

53 Cryptoprocta ferox Cryptoprocta ferox Fossa fossana Fossa fossana 79 98 Galidia elegans (C) 99 75 Galidia elegans (C) Galidia elegans (N) 100 Galidia elegans (S) 99 100 98 Galidictis fasciata Galidia elegans (N) 59 Mungotictis decemlineata 100 100 Galidictis fasciata Crossarchus obscurus Mungotictis decemlineata 93 Suricata suricatta Crossarchus obscurus Cynictis pencillata 89 Mungos mungo 100 100 Cynictis pencillata 83 100 97 Herpestes edwardsii Herpestes javanicus 100 Herpestes edwardsii 100 Herpestes javanicus Suricata suricatta 59 79 Mungos mungo Crocuta crocuta Crocuta crocuta 100 Civettictis civetta 100 Civettictis civetta 97 Viverra tangalunga 77 79 Viverra tangalunga 97 Genetta servalina 99 100 Genetta servalina 100 Hemigalus derbyanus 92 Hemigalus derbyanus Paradoxurus 96 100 Paradoxurus Felis silvestris 100 Felis silvestris 50 Panthera leo 100 Panthera leo Nandinia binotata Nandinia binotata 96 69 Procyon lotor Canis lupus 100 100 Erignathus barbatus 77 Tremarctos ornatus Tremarctos ornatus 92 Procyon lotor Canis lupus Erignathus barbatus Scalopus Scalopus Homo Homo