Miniopterus mahafaliensis, Goodman & Maminirina & Bradman & Christidis & Appleton, 2009

Goodman, Steven M., Maminirina, Claudette P., Bradman, Helen M., Christidis, Les & Appleton, Belinda, 2009, The Use of Molecular Phylogenetic and Morphological Tools to Identify Cryptic and Paraphyletic Species: Examples from the Diminutive Long-fingered Bats (Chiroptera: Miniopteridae: Miniopterus) on Madagascar, American Museum Novitates 3669, pp. 1-36 : 17-28

Miniopterus mahafaliensis , new species

Figures 4D–E , 6 , 7 ; tables 3–5

HOLOTYPE: Field Museum of Natural History ( FMNH) 173197 collected by S.M. Goodman (field number SMG 12649) on 2 March 2002. The skull is in fine condition. Before preservation of the specimen in formaldehyde, the skull was removed, placed in dilute ethanol, and then cleaned using dermestid beetles. Pectoral muscle samples saved in lysis buffer; this animal was sequenced and was used in the phylogenetic analysis present- ed herein (fig. 2; table 1). The specimen has a full adult dentition and the basisphenoidbasioccipital sutures are completely fused.

Measurements are in mm and body mass in g. Total length, 95; tail length, 44; hind foot length, 6; tragus length, 6; ear length, 11; forearm length, 38; mass, 5.3; greatest skull length, 13.7; greatest zygomatic breadth, 7.0; postorbital breadth, 3.0; mastoid breadth, 7.0; greatest braincase width, 6.8; lachrymal width, 3.5; palatal length, 6.3; mandible length, 9.5; cranial toothrow, 6.0; upper canine-molar toothrow, 5.1; width across upper canines, 3.7; width across third upper molars, 5.0 (tables 3–5).

TYPE LOCALITY: Madagascar: Province de Toliara, Parc National de Tsimanampetsotsa, 6.5 km NE Efoetse, near Mitoho Cave , 24 ° 03.09S, 43 ° 45.09E, at 50 m above sea level .

DIAGNOSIS: A diminutive species of Miniopterus with relatively dense pelage and medium-brown dorsum and light gray-tipped ventrum. Uropatagium with relatively dense fur on the dorsal surface. The holotype forearm length is 38 mm and in the type series ranges from 35–40 mm (mean 5 37.4 mm). Tragus is relatively long, 6 mm in the holotype and in the type specimens ranges from 5– 6 mm (mean 5 5.8 mm). The tragus is moderately wide, has parallel margins along most of its length, and the distal portion curves medially into a slightly expanded and rounded tip. The cranium has a prominent sagittal crest in contact with the parietals, relatively short and wide nasal sulcus, and deep cut V-shaped palatal emargination.

PARATYPES: Province de Fianarantsoa, Parc National de l’Isalo, along Sahanafa River, at foot of Bevato , 28 km SE Berenty- Betsileo , 22 ° 19.09S, 45 ° 17.69E, 550 m, 9 December 2002 ( FMNH 175991 ) ; Ihosy, commune rurale d’Akily, Grotte d’Andranomilitry, 22 ° 23.1119S, 46 ° 03.3559E, 950 m, 1 November 2004 ( FMNH 184470–184473 ) . Province de Toliara, Parc National de Kirindy-Mite , 13 km W Marofihitsa, 20 ° 47.49S, 44 ° 08.89E, 30 m, 11 November 2002 ( FMNH 176096–176099 ) ; Parc National de Kirindy-Mite , near village of Betakilotra, 11 km SE Marofihitsa, 20 ° 53.29S, 44 ° 04.89E, 35 m, 16–17 November 2002 ( FMNH 176100–176110 ) ; Forêt des Mikea , 9.5 km W Ankililoaka, 22 ° 46.79S, 43 ° 31.49E, 80 m, 14, 15, and 18 February 2003 ( FMNH 176167–176170 ) ; Ranobe, 23 ° 14.0339S, 43 ° 52.4939E, approximately 50 m, 16 August 2003 ( FMNH 177376 ) ; Fiherenana, 23 ° 149170S, 43 ° 529230E, 27 November 2002 ( FMNH 176509 , 176510 ) ; Betroka, 23 ° 159500S, 46 ° 059300E, 800 m, 18 June 1953 ( FMNH 75774 ) ; Antafiky, 23 ° 29916.00S, 044 ° 04939.10E, 50 m, 24 July 2001, 4 February 2002 ( FMNH 173252–173253 ) ; Grotte d’Ambanilia, 3.7 km SSE Sarodrano , 23 ° 32.3979S, 43 ° 44.7639E, sea level, 7 May 2002 ( FMNH 172918–172928 ) ; Grotte de Bisihiko , 0.75 km E St. Augustin, 23 ° 32.9339S, 43 ° 46.0449E, 10 m, 8 May 2002 ( FMNH 172929–172933 , 202484–202485 ) ; Mahaleotse, 23 ° 31938.80S, 44 ° 05916.10E, 70 m, 4 June 2002 ( FMNH 176508 ) ; Parc National de Tsimanampetsotsa , Malaza Manga Aven, 24 ° 1.8279S, 43 ° 45.2839E, 80 m, 4 March 2002 ( FMNH 173204 ) ; Parc National de Tsimanampetsotsa, 6.5 km NE Efoetse, near Mitoho Cave , 24 ° 03.09S, 43 ° 45.09E, 50 m, 28 February 2002, 2 March 2002 ( FMNH 173154 , 173189–173196 , 173198– 173203 ; same locality as the holotype) ; Grotte d’Antagneotsy, 5.0 km NE Vohombe, 24 ° 23.0019S, 43 ° 50.7429E, 100 m, 26 February 2005 ( FMNH 184226 ) ; Grotte d’Amborombe, 4.0 km Itampolo , 24 ° 37942.70S, 43 ° 58956.90E, 70 m, 29 May 2005 ( FMNH 184160 ) ; Grotte d’Andraimpano, 4.2 km NE Itampolo , 24 ° 39.0129S, 43 ° 57.7979E, 110 m, 22–23 February 2005, 28 May 2005 ( FMNH 184155–184159 , 184168 , 184222–184225 ) ; 10.5 km SE Itampolo (village), 24 ° 44.29S, 44 ° 1.799E, 120 m, 18 February 2005 ( FMNH 184217–184221 ) .

DISTRIBUTION: Miniopterus mahafaliensis is known from numerous sites on or in close proximity to the limestone Mahafaly Plateau in the southwestern portion of the island, including the zone from Itampolo north across the Onilahy River to Sarodrano spanning the elevational range from sea level to 120 m (fig. 1). Its distribution then continues further north to the Forêt des Mikea (70–80 m) and based on current data ends in the Kirindy-Mite region (30–35 m). Further, it is known from the more inland and upland sites of Ihosy and near Betroka from 800–950 m and within the Isalo formation at 550 m.

DESCRIPTION: A small species of Miniopterus with the tail length less than one-half the total length (table 3). Forearm length ranging in the type series from 35–40 mm and the hind foot length from 6–7 mm. In the holotype and associated type series, the body fur is relatively long and dense, and the dorsum coloration is a medium brown and the ventrum hairs are tipped with a light gray, giving a notably lighter appearance to the underside (fig. 6). Wing membranes are medium brown, grading into a slightly lighter brown in the uropatagium. This latter membrane has a relatively dense covering of hair across its surface, being more obvious on the dorsal side toward the proximal half of the membrane and sparser on the ventral side. Uropatagium and plagiopatagium attaching to the femur at a position superior to the ankle joint.

On the dorsal surface of the ring-shaped ears, the proximal one-half has a covering of hair across much of its surface and the distal half is sparsely furred. In contrast, the ventral surface is sparsely covered with fur, being concentrated around the distal ear margin. The ear terminates with a slightly elongated tip, rounded anteriomedially. The average tragus length in the holotype and other animals in the type series is 5.8 mm (range 5–6 mm) (table 3). The proximal three-quarters of the moderately wide tragus have a shaft with largely parallel sides, distally curving slightly medially, and expanding into a round- ed tip (fig. 4D–4E). In some individuals, there is a lateral flange along the proximal threequarters of the tragus.

The skull of M. mahafaliensis has a slightly elongated rostrum, slightly bulbous braincase, and not notably constricted at the level of the interorbitals (fig. 7). In dorsal view, the rostrum is relatively linear in shape, particularly the nasals, which do not show a marked lateral expansion in their medial portions. The central sulcus of the nasal region is relatively narrow and spanning, in most cases, slightly more than the proximal one-half of the nasal length. Frontals somewhat rounded with prominent sagittal crest passing distally to the parietals; in lateral view, there is a slight flattening of the cranium, without a notable depression, in the parietal region. Lambdoid crest is prominent. When viewed dorsally, the palatal emargination forms a deeply cut Vshaped notch. Medial portion of palate is slightly concave with lateral lingual sides of the toothrows forming two elongated and parallel surfaces, and posterior palatal spine relatively long and thin. In most individuals, the palatal foramina, located at the level of the last upper molar, are indistinct or not present.

Dental formula I 2/3 C 1/1 PM 2/3 M 3/3, comprising the adult dentition of 36 teeth, and characteristic of Miniopterus ( Koopman, 1994) . The dentition of M. mahafaliensis is typical of small members of this genus, with, for example, the first premolar (PM2) being relatively small and with simpler cusp morphology than the second premolar (PM4). In lateral view, the length of PM4 is approximately two-thirds that of the C.

On the basis of t-test comparisons, four of the 19 measured variables for M. mahafaliensis demonstrated evidence of sexual dimorphism. Among the external measurements listed in table 3, only mass showed a difference between the sexes—on average 5.0 g in females, several of which were pregnant, and 4.7 g in males. None of the cranial measurements demonstrated sexual dimorphism. In contrast, three of the four dental measurements were sexually dimorphic: cranial toothrow, in males 6.0 mm and females 5.9 mm (t 5 2.00, P 5 0.05, df 5 71); upper canine-molar toothrow, in males 5.0 mm and females 4.9 mm (t 5 2.07, P 5 0.04, df 5 71); and width across upper canines, in males 3.6 mm and females 3.5 mm (t 5 4.21, P 5 0.0001, df 5 71). While statistically significant, in part based on the large sample sizes, the differences between the sexes are rather subtle and we consider them unimportant with regard to the analyses presented herein and the sexes have been combined.

MORPHOLOGICAL AND MORPHOMETRIC COM- PARISONS: There are at least six species of diminutive Miniopterus within the Malagasy region ( Madagascar and the Comoros Archipelago) ( Goodman et al., 2008, 2009b; Weyeneth et al., 2008): M. petersoni (average forearm length of 39.8 mm, range 38–43 mm); M. manavi sensu stricto (average forearm length of 38.6 mm, range 38–39 mm); M. griveaudi (average forearm length of 36.9 mm, range 35–38 mm); M. aelleni (average forearm length of 38.8 mm, range 35– 41 mm); M. brachytragos (average forearm length of 36.6 mm, range 35–38 mm); and M. mahafaliensis (average forearm length of 37.4 mm, range 35–38 mm) (all of these measurements were made by the same collector, with the exception of M. manavi ). Of these six species, four are endemic to Madagascar and the other two ( M. aelleni and M. griveaudi ) restricted to Madagascar, Anjouan, and Grande Comore. These species can be differentiated at the first level based on molecular genetic data (fig. 2) and differences in tragus shape (fig. 4; fig. 3 in Goodman et al., 2009b), and at a second level using a combination of other external and craniodental characters and morphology.

The only diminutive East African member of this genus, M. minor , has been shown to be genetically separate from the Malagasy species (fig. 2; Goodman et al., 2009b; Weyeneth et al., 2008) and does not need to be considered further. Several Indomalayan, Australian, and Oceanian taxa fall into the same size range as the Malagasy animals, but based on geographic arguments can be removed from evaluation. Hence, species necessary to demonstrate the distinctiveness of M. brachytragos and M. mahafaliensis are all restricted to the Malagasy region. Given its slightly larger size, M. petersoni can also be eliminated from these comparisons ( Goodman et al., 2008).

Miniopterus brachytragos can be distinguished from the other regional members of this genus based on tragus shape, which is notably short and blunt, as well as being the only one species with sparse hairs occurring on this structure (fig. 4A–C). Further, M. mahafaliensis has a tragus morphology that separates it from the other four diminutive Malagasy Miniopterus spp. , and can be possibly confused only with M. griveaudi and M. aelleni . The tragus in M. mahafaliensis is a moderately thick structure with two largely parallel sides that terminate distally with a slightly expanded rounded tip that curves medially (fig. 4D–E); M. griveaudi has a notably thinner tragus that has a relatively straight shaft and the structure terminates with a slightly retracted and rounded head (fig. 4F); M. aelleni has a thickset tragus, distinctly wider at the base, that tapers toward the distal slightly pointed tip (fig. 4G); and M. manavi has a relatively thin tragus and the medial margin along the distal two-thirds of its length has a flange, the distal lateral portion is slightly enlarged and rounded, and the distal medial tip terminates with an angular, straight edge (fig. 3a in Goodman et al., 2009b). In some photographic images we have examined of agitated handheld animals, the tragus is bent anteriorly compared to fluid-preserved specimens; this aspect needs to be taken into consideration when making comparisons to fig. 4.

Of these five taxa, M. manavi has the darkest dorsal pelage coloration, tending toward black or a slightly lighter dark brownish black. The fur of M. petersoni , M. griveaudi , and M. aelleni , particularly the dorsum, have different tints of dark brown and in a few adults approach reddish brown, while in M. mahafaliensis and M. brachytragos the dorsum tends to be a lighter medium brown. In all five species, the venter is paler in coloration than the dorsum, specifically on the distal portions of the hairs, giving a slightly mottled appearance; the exception is M. mahafaliensis , which is distinctly grizzled. The wing and tail membrane coloration in M. manavi is largely a uniform dark brownishblack, while these surfaces in the remaining four taxa are generally dark to medium brown on the wings, grading into a lighter brown on the interfemoral membrane. The amount of fur covering the uropatagium also helps to distinguish these species: in M. manavi , M. mahafaliensis , and M. brachytragos there is relatively dense fur over more than half the proximal dorsal surface and more sparse on the proximal ventral surface; in M. aelleni there is thin fur across the surface of this membrane, being more obvious on the dorsal than ventral portions; and in M. griveaudi the membrane is largely naked, with the exception of thin hairs often difficult to see with the naked eye.

There are a number of cranial characters that help to separate these five taxa and here we restrict comparisons to the most obvious features. In general, from dorsal view, the rostrum of M. aelleni is linear in shape and distinctly longer than M. brachytragos , M. griveaudi , M. mahafaliensis , and M. manavi , which are characterized by reduced lateral expansion on the medial portion of nasals (fig. 8). Miniopterus griveaudi tends to have an even more rounded rostrum than M. manavi . In M. aelleni , M. griveaudi , and M. mahafaliensis the sagittal crest passes distally to the parietal region, while in M. brachytragos and M. manavi it extends two-thirds of the distance to the parietals. The lambdoid crest in M. aelleni , M. griveaudi , and M. mahafaliensis is more prominent than in the remaining two taxa. The palatal emargination in M. aelleni forms a deeply cut V-shaped notch, in M. griveaudi , M. mahafaliensis , and M. manavi this is a more open V-shape, and in M. brachytragos a notably open and proximally rounded U shape.

The medial portion of the palate in M. aelleni and M. manavi is notably flat, while in M. brachytragos , M. griveaudi , and M. mahafaliensis the palate forms a slightly curved basin (fig. 9). In all five taxa, the basisphenoid pits are shallow and narrow in shape, with some subtle variation between them and a tendency to be more elongated in M. manavi . If palatal foramina are present, they tend to be more regular in M. brachytragos and are located at the level of the last upper molar. In general, the dentitions of these five species are similar (figs. 9, 10).

Principal component analyses were conduct- ed separately on cranial and dental measurements of the five species that had been classically placed under the name manavi : M. aelleni , M. brachytragos , M. griveaudi , M. mahafaliensis , and M. manavi sensu stricto, as well as the slightly larger M. petersoni . When factors 1 and 2 are plotted for the cranial variables (fig. 11A), individuals of M. petersoni show a clear separation from the balance of the taxa, including M. manavi sensu stricto, individuals of M. aelleni form largely a separate group, and M. griveaudi and M. brachytragos show no overlap. Certain taxa ( M. brachytragos , M. griveaudi , M. mahafaliensis , and M. manavi sensu stricto) show broad overlap in their spatial positions, which emphasizes the similar cranial size and morphology. All of the cranial variables showed heavy loadings on the first factor, which accounted for 71.5% of the explained variation (table 6). On the second factor, no variable displayed an important loading score and an additional 13.2% of the variation was explained. In general, M. petersoni and M. aelleni have relatively long and narrow skulls and, at the opposite extreme, M. griveaudi short and wide skulls.

The results of the principal component analysis for the dental variables (fig. 11B) show the same basic distribution for the plot of factors 1 and 2 as with the cranial variables. The exceptions include one individual of M. petersoni that falls within the multispecies cluster. Based on this analysis, M. brachytragos , M. griveaudi , M. mahafaliensis , and M. manavi sensu stricto, show considerable similarity in dental size and shape. Of the four variables used in this analysis, all showed heavy loadings on factor 1, which explains 81.7% of the variance (table 6). With the addition of factor 2, and additional 11.7% of the variance is explained and not one of the variables demonstrated heavy loading.

TABLE 6 Factor loadings from principal component analysis of cranial and dental measurements of specimens of Miniopterus manavi , M. griveaudi , M. aelleni , M. mafaliensis , M. petersoni , and M. brachytragos The measurement greatest zygomatic breadth (ZYGO) has been removed from the analysis to augment sample sizes. A graphical representation of the first two factors is presented separately for cranial and dental variables in fig. 11. See Methods for an explanation of variable acronyms.

PHYLOGEOGRAPHY: Miniopterus mahafaliensis has an intraclade divergence of 2.2% K 2P. Among the sequenced animals, there appears to be no clear phylogeographic structure, as, for example, among animals from Kirindy-Mite, which are distributed among the different subclades (fig. 2).

ECOLOGICAL NOTES: The specimens of M. mahafaliensis captured at the holotype site, near Mitoho Cave in the PN de Tsimanampetsotsa, were in largely undisturbed southwestern dry spiny forest-thicket resting on exposed limestone at the edge of the Mahafaly Plateau. The series was obtained in mist nets erected near the Grotte d’Andranoilovy, which is less than 100 m from the entrance of the Mitoho Cave; these two caves open horizontally and are presumed to be part of the same complex. At the nearby Malaza Manga Aven, in similar habitat, a single individual was captured in a harp trap as it exited a relatively deep sinkhole after dusk. A number of other individuals of this taxon were obtained in similar limestone cave settings on the Mahafaly Plateau to the south of Mitoho Cave, including the Grotte d’Antagneotsy, Grotte d’Amborombe, and Grotte d’Andraimpano, and to the north at the sea cave of Ambanilia and in the coastal littoral at the Grotte de Bisihiko. The habitat surrounding these different cave sites ranged from relatively undisturbed to heavily disturbed southwestern dry spiny forest-thicket and southwestern coastal bushland habitats.

Further to the north and away from any large blocks of exposed sedimentary rock, but still in lowland habitat, M. mahafaliensis is known from a site in the Forêt des Mikea in disturbed transitional southwestern dry spiny forest-thicket/western dry forest resting on red sand substrate. Several individuals were captured further to the north in the Parc National de Kirindy-Mite in slightly disturbed western dry forest. At a site near the village of Betakilotra and within the same park, several animals were netted as they descended into a narrow dug well shaft, presumably to drink water. This latter locality was in heavily degraded mixed open habitat and secondary dry deciduous forest.

Miniopterus mahafaliensis is known from two inland and upland central southwest sites with eroded sandstone outcrops. It was captured in the Parc National de l’Isalo in a mist net traversing the Sahanafa River, a zone with degraded gallery forest dominated by introduced mangos and within a few hundred meters from exposed sandstone cliffs. The second sedimentary rock site is the Grotte d’Andranomilitry, near Ihosy, is a shallow cave with a small river passing through the interior and surrounded by open savannah. An older specimen was obtained near Betroka and we have no specific details on the collection site.

ETYMOLOGY: The name mahafaliensis is derived from the Malagasy word mahafaly, meaning ‘‘to make taboos,’’ but here specifically referring to one of the local cultural groups in southwestern Madagascar, the Mahafaly, and the Mahafaly Plateau, a limestone karst area, from whence many of the specimens of this taxon were collected. In the region of the Forêt des Mikea, the Malagasy name for this bat was given by a local hunter as kitrotroke.

Figures

Fig. 4. Right ear and tragus of Miniopterus spp. from Madagascar: A, M. brachytragos (FMNH 175840, holotype), from Parc National de Namoroka, Forêt d’Ambovonomby, 26 km NW Andranomavo; B, M. brachytragos (FMNH 172685) from the Forêt de Binara, near Analamazava River, 7.5 km SW Daraina (village); C, M. brachytragos (FMNH 202523) from 1.8 km S Ambanizana, Masoala Peninsula; D, M. mahafaliensis (FMNH 173197, holotype) from Parc National de Tsimanampetsotsa, 6.5 km NE Efoetse, near Mitoho Cave; E, M. mahafaliensis (FMNH 176101) from Parc National de Kirindy-Mite, near village of Betakilotra, 11 km SE Marofihitsa; F, M. griveaudi (FMNH 184055) from Beloboka, grotte n°1, 12.5 km E Mahajanga, Madagascar; G, M. aelleni (FMNH 175842) from the Parc National de Namoroka, Forêt d’Ambovonomby, 26 km NW Andranomavo. The slightly elongated and pointed distal portion of the tragus in FMNH 175840 (A), as compared to FMNH 172685 and FMNH 202523, maybe an anomalous shape in M. brachytragos.

Fig. 6. Photograph of living Miniopterus mahafaliensis captured 6.5 km NE Efoetse, near Mitoho Cave, Parc National de Tsimanampetsotsa (FMNH 173191), taken at the same locality as the holotype (FMNH 173197). Note the gray grizzled hair to the ventrum. (Photograph by Harald Schütz.)

Fig. 7. Different views of skull and mandible of the holotype of Miniopterus mahafaliensis (FMNH 173197) collected in the Parc National de Tsimanampetsotsa, 6.5 km NE Efoetse, near Mitoho Cave: (above, left) dorsal view of cranium; (above, right) ventral view of cranium; and (below) lateral view of cranium and mandible. (Photograph taken by John Weinstein, Field Museum image number Z94487_05d.)