We describe a new species of Varanus from Mussau Island, north-east of New Guinea. The new species is a member of the Varanus indicus species group and is distinguished from all other members by both morphological and molecular genetic characters. It is the third species of Varanus reported from the Bismarck Archipelago and the first record of a yellow tongued member of the Varanus indicus species group from a remote oceanic island. The herpetofauna of Mussau Island has not been well studied but the discovery of this new species is in accordance with recent findings indicating that the island may harbor several unknown endemic vertebrates. The distribution of the closely related Varanus finschi is also discussed in the light of recent fieldwork and a review of old records.

Melanesia, Bismarck Archipelago, St. Matthias islands, Varanidae , Varanus doreanus , Varanus finschi , Varanus yuwonoi , mitochondrial phylogeny, biogeography, taxonomy

The varanid subgenus Euprepiosaurus Fitzinger comprises two species groups: the V. indicus and V. prasinus species groups. The subgenus is geographically restricted to a large region east of Wallace’s line with the Solomon Islands and parts of Micronesia forming the eastern and northern boundaries (Ziegler et al. 2007a, Sweet and Pianka 2007). The systematic arrangement is well-supported by molecular and morphological studies (Ziegler and Böhme 1997, Fitch et al. 2006, Vidal et al. 2012). Several new monitor lizards of the subgenus Euprepiosaurus have been discovered from islands in the southwest Pacific since the early 1990s. This increase has mainly been the result of taxonomic studies of museum collections, and the appearance of novel species through the international trade in live animals. Eleven species have been described from the Moluccas and Raja Ampat islands in eastern Indonesia, more often as a result of new specimens arriving through the animal trade rather than resulting from field studies and scientific collections (e.g. Böhme and Ziegler 1997, Harvey and Barker 1998).

Over the same time period, the monitors of Papua New Guinea and the Solomon Islands have received considerably less scientific attention. Papua New Guinea has no legal international live animal trade and it’s fauna is less represented in European museum collections. Since 1990 only two new species have been described from Papua New Guinea, both from revisions of colonial era museum collections: Varanus telenesetes (Sprackland 1991) (possibly a synonym of V. bogerti Mertens [Weijola obs.]), and Varanus finschi Böhme, Horn & Ziegler, 1994. As a consequence, the Melanesian islands have been considered less diverse in comparison to the Moluccas (Ziegler et al. 2007a).

As part of a larger survey of the monitors of the Bismarck Archipelago of Papua New Guinea in 2012, VW collected three specimens of a previously unknown blue-tailed species of the V. indicus species group from Mussau Island in the St Matthias group. Previously three individual monitor lizards in total had been recorded on two separate occasions from the St Matthias group - a juvenile specimen collected in 1944 (AMNH 85887) and two adult specimens collected during the Noona Dan Expedition in 1961-1962 (ZMUC 4272-4273) (identified as V. finschi in Philipp et al. [2007]).

Four species of the V. indicus species group (including the new taxon from Mussau) share the occurrence of yellow pigmentation on the tongue (Harvey and Barker 1998). Although taxon sampling in published molecular phylogenies has been limited, these yellow-tongued monitors have consistently formed a basal clade within the V. indicus species group (Ast 2001, Welton et al. 2013). Varanus doreanus Meyer is widespread on New Guinea, Aru, Biak, Waigeo, Salawati and parts of northern Cape York (Ziegler et al. 2007a). Varanus finschi Böhme, Horn & Ziegler, 1994 is likely endemic to New Britain (see discussion). Varanus yuwonoi Harvey & Barker is endemic to Halmahera and possibly nearby islands (Weijola 2010).

Molecular genetic and morphological studies of the newly collected material from Mussau Island clearly show the population represents a distinct taxon of yellow-tongued monitor. The concept best applicable to allopatric species is probably the Evolutionary Species Concept (ESC) (Simpson 1951) and more recent integrative approaches such as the Unified Species Concept (de Queiroz 2007). On account of its distinctive morphology, phylogenetic position and geographically isolated distribution we recognize the Mussau monitor as a unique evolutionary lineage and describe it as a new species herein.

Taxonomy. We follow the nomenclature of de Lisle (2009) for the taxa treated. The taxonomic identities of V. cerambonensis and V. indicus (sensu Philipp et al. 1999) included in the molecular phylogeny have recently been challenged (see Weijola and Sweet 2015, Weijola 2015) but until a ruling from the ICZN is issued we follow the nomenclature of Philipp et al. (1999).

Morphology. We obtained data for the meristic characters used by Brandenburg (1983) and in later works on the V. indicus group (e.g., Ziegler et al. 2007b, Weijola and Sweet 2010). Measurements were taken to the nearest 0.5 mm (head) or 1 mm with a steel tape or calipers. Comparative scale counts for V. doreanus and V. yuwonoi were taken from the literature (Brandenburg 1983, Harvey and Barker 1998, Ziegler et al. 2007b). Specimens listed in Brandenburg (1983) were identified by VW. We used PAST (Hammer et al. 2001) for Principal Components Analyses (PCA). The variance-covariance matrix was used on the unaltered scalation data including P, Q, S, T, X, XY, m, N and R characters. Definitions of, and abbreviations used for measurements, proportion indices and scale counts are presented in Table 1.

Museum abbreviations used are: ABTC: Australian Biological Tissue Collection (South Australian Museum, Adelaide), AMNH: American Museum of Natural History (New York), AMS: Australian Museum (Sydney), BPBM: Bernice Pauahi Bishop Museum (Honolulu), NMW: Naturhistorische Museum Wien (Wien), QM: Queensland Museum, RMNH: Naturalis museum (Leiden), UMMZ: Museum of Zoology, University of Michigan, ZMA: Zoological Museum of the University of Amsterdam (currently Naturalis), ZMB: Zoologische Museum der Humboldt Universität (Berlin), ZMUC: Zoological Museum, University of Copenhagen, and ZMUT: Zoological Museum, University of Turku.

Molecular genetic methods. A 661 bp fragment of the mitochondrial genome, including the 3’ end of the NADH dehydrogenase subunit 4 (ND4) gene (710 bp) and the 5’ end of tRNA^His (64 bp) gene, was amplified and sequenced (hereafter referred as ND4) using the forward primer 5’- TGA CTA CCA AAA GCT CAT GTA GAA GC-3’ (Forstner et al. 1995) with the reverse primer 5’ CAT TAC TTT TTA CTT GGA TTT GCA CCA-3’ (Arévalo et al. 1994). A 566 bp fragment of the mitochondrial 16S rRNA gene was amplified and sequenced using the forward primer: 5’ - CGC CTG TTT ATC AAA AAC AT - 3’ with the reverse primer: 5’ - CCG GTC TGA ACT CAG ATC ACG T – 3’ (Palumbi et al. 1991).

The amplification reactions were performed in a final volume of 50ul using the Phusion U Hot Start PCR Master Mix (ThermoFisher Scientific, St. Leon-Rot, Germany). The PCR profile for the ND4 amplification was 9 min at 94 °C (initialization step, one cycle), 30 sec at 94 °C (denaturation step, 35 cycles), 25 sec at 46,5 °C (annealing step, 35 cycles), 35 sec at 72 °C (extension step, 35 cycles) and 2 min at 72 °C (final elongation step, 1 cycle). The corresponding profile for the 16S rRNA amplification was 9 min at 94 °C (initialization step, one cycle), 30 sec at 94 °C (denaturation step, 35 cycles), 25 sec at 55 °C (annealing step, 35 cycles), 35 sec at 72 °C (extension step, 35 cycles) and 2 min at 72 °C (final elongation step, 1 cycle). A negative control (no template present) was also included in all PCRs. All PCR products were analyzed by gel electrophoresis on a 1.8% agarose gel containing 0.5 µg/ml ethidium bromide (Promega, Madison, USA) before they were sequenced.

PCR products were sequenced by the Beckman Coulter Genomics company (Essex, UK). GenBank accession numbers of the new sequences are provided in Table 2.

Phylogenetic analysis. Resulting sequences were aligned by MUSCLE (Edgar 2004) as implemented in GENEIOUS v8.1.4 and concatenated for phylogenetic analysis. Bayes factors were used to assess all possible alternative partitioning strategies for five data subsets: 1st, 2nd and 3rd codon positions, the tRNA and 16S rRNA in PartitionFinder v1.0.0 (Lanfear et al. 2012). The Akaike Information Criterion (AIC) and Bayes Information Criterion (BIC) were used to assess the best fit partition strategy and nucleotide substitution model for each data subset in the selected partition strategy. Sequences were analysed phylogenetically using Bayesian and maximum likelihood (ML) methods. Bayesian analysis was conducted using MrBayes v3.2.5 (Ronquist and Huelsenbeck 2003). The analysis was run with model parameters unlinked using default priors for ten million generations with two independent runs and two chains sampling every 500 generations. The first 25% of sampled trees were discarded as burn-in and convergence was assessed by examining effective sample sizes (ESS values), split frequencies of clades across runs and likelihood plots through time in TRACER v1.6 (Rambaut and Drummond 2007). Evolutionary trees were constructed with the ML criterion of optimality implemented in the web server version of RAxML (Stamatakis et al. 2008), which uses the GTR+Γ model of nucleotide substitution. The robustness of phylogenetic hypotheses was tested with non-parametric bootstrapping. Varanus prasinus, from the sister lineage to the V. indicus species group, was used as outgroup.

Net average sequence divergence between lineages (dA) was calculated from the ND4 data only in MEGA v5 (Tamura et al. 2011) as: dA = dXY – (dX + dY)/2, where, dXY is the average distance between groups X and Y, and dX and dY are the within-group means. Net average sequence divergence was calculated more broadly for sister species pairs of Varanus where more than one sequence was available for each member of the pair from our data and the data of Fitch et al. (2006), Smith et al. (2007), Smissen et al. (2013), Maryan et al. (2014), Doughty et al. (2014) and GenBank accessions for V. komodoensis.

Biogeography. The members of the V. indicus species group have been extraordinarily successful at colonizing the islands of the SW Pacific. Varanus indicus and its closest relatives, which are adept at oversea dispersal, have reached most islands between the western Moluccas and eastern Solomon islands. The yellow-tongued monitors on the other hand have, been far less adept at oversea dispersal. Varanus doreanus populations are with few exceptions (such as Biak) restricted to the land bridge islands of New Guinea. Varanus yuwonoi to Halmahera, a geologically complex island which was much more closely associated with parts of western New Guinea during the Miocene and Pliocene (Hall 1998) when it may have been easier to colonize by monitors and other terrestrial animals. Varanus finschi likely reached the nearby New Britain through oversea dispersal as this island has no known historical landbridges to New Guinea. Varanus semotus is notable as it is separated from its closest relatives by hundreds of kilometers of open sea and must have colonized the oceanic Mussau Island through long distance oversea dispersal, most likely by rafting. Vidal et al. (2012) estimate the age of V. indicus species group at around 6–11.5 mya. With this time reference the subsequent lineage diversification of species group should have occurred sometime in the late Miocene to early Pleistocene during which it is also likely that Mussau was colonized.

The St. Matthias group is situated on northern arc of the Bismarck Archipelago and has never had land connections to larger landmasses. It has three known endemic species of passerine birds; the Mussau monarch (Symposiachrus menckei), the Mussau triller (Lalage conjuncta) and the Mussau fantail (Rhipidura matthiae), but this number was most likely greater prior to human colonization (Steadman and Kirch 1998). There are no known native terrestrial mammals on Mussau but three still undescribed species of bats have recently been discovered (Flannery 1995, Aplin et al. 2015). Very little has been published on the herpetofauna of Mussau (e.g. Brown 1955, Mys 1988, Richards and Aplin 2015) and most of the recorded species are either widespread tramp species or endemics shared by Mussau and Manus. A recent (2014) faunal survey conducted by the Wildlife Conservation Society discovered a new endemic species of frog of the genus Cornufer (which constitute half of the known amphibian fauna). All nine species of skinks (single species of Carlia, Eugongylus, Lamprolepis, Lipinia and Sphenomorphus and 4 species of Emoia) recorded by the same expedition are widespread while one of the four species (2 Gehyra, 1 Gekko and 1 Nactus) of gekko (Gehyra sp.) is reported to be a new species endemic to Manus and Mussau Island (Richards and Aplin 2015). According to Richards and Aplin (2015) it is likely that additional species occur in the still unexplored fragments of primary forest of the interior. For now V. semotus is the only endemic lizard known from Mussau.

The absence of Varanus indicus s.l. which is otherwise almost universal on islands in the Southwest Pacific, including Manus and New Hanover, is more difficult to explain. The lack of widespread mangrove swamps around the coastlines seems an insufficient explanation as most island populations of Mangrove monitors are habitat generalists that occur in various coastal and inland habitat types (Weijola and Sweet 2015).

Varanus finschi. Virtually nothing has been published on the biology of V. finschi since its initial description over two decades ago. In 1988 SCD collected a specimen at Amelei on the south coast of New Britain (AMS 129614). In 2012 VW visually identified four and collected two specimens in the vicinity of Rabaul, Kokopo and Nodup at the northern end of East New Britain (ZMUT Sa186 & 190). These new samples allowed us to include the species in a larger molecular phylogeny of the V. indicus group for the first time. The samples of alleged V. finschi (BPBM 17250 & 19510) from Milne Bay Province used by Ziegler et al. (2007) were re-identified as V. cf. jobiensis (by VW). Examination of live specimens also showed that the tongue color of V. finschi is yellow rather than pink/light as reported earlier (Sprackland 1997, Harvey and Barker 1998). According to VW’s field observations V. finschi is most numerous along the coast. Attempts to find monitors higher up in the Baining Mountains (500-700 m. elev.) were unsuccessful despite local testimonies of occasional observations. Varanus indicus is common along the coast and in the mangroves of New Britain and there appears to be at least partial habitat overlap between the two species.

Varanus finschi has been reported to have an extensive range outside of New Britain including New Ireland, New Guinea (Ziegler et al. 1999), northern Australia (Ziegler et al. 2001) and the Kei Islands (Philipp et al. 2004). However, as the only records from New Guinea (ZMB 18838 & 18839) and Queensland (NMW 12329-6 & 12429-8) are based on colonial-era museum vouchers without detailed collection information we consider them unreliable. The records for the Kei islands and New Ireland stem from misidentification of populations of V. cf. indicus with high scalecounts, pink tongue and similar dorsal pattern to V. finschi (Weijola pers. obs.). There is a single record from the Duke of York Islands (AMS R5618) but VW was not able to verify its occurrence there during a field survey in 2012. Thus, as far as we are aware, all verifiable records of V. finschi are from New Britain.

Conservation. The field observations indicate that V. semotus doesn’t occur, or possibly only at low densities, in the highly degraded secondary forest/bush of large parts of the interior of the island. It is likely that the species occurred throughout Mussau prior to the large scale logging activities of the past three decades (Venter and Arihafa 2015). Thus the species is now mostly restricted to the coastal strip of a relatively small isolated island. Possible threats to the future survival of this species would be the introduction of cane toads which were widely established in the PNG islands during WW2 (Zug et al. 1975). According to unconfirmed accounts by locals they already occur on Emirau Island which also according to local inhabitants on Mussau lack monitor lizards. Varanus semotus is the only large-sized terrestrial generalist predator and scavenger on the island, and may well fill an important ecological function, making it of particular conservation concern. The new species is unusual inasmuch as it fills a role normally occupied by Mangrove monitors on isolated Pacific islands and it can well be considered a biogeographical oddity.

We thank the following people and institutions in Port Moresby, Papua New Guinea for providing research permits and facilitating Weijolas fieldwork: Georgia Kaipu (National Research Institute), Barnabas Wilmot (Department of Environment and Conservation), Bulisa Iova, Ilaiah Bigilale (National Museum and Arts Gallery) and Ralph Mana (University of Papua New Guinea). Weijola is also grateful to the provincial government of New Ireland for providing a local research permit. Field expenses were covered by grants from Svensk-Österbottniska samfundet, Svenska Kulturfonden, Nordenskiöld-Samfundet, Turun Yliopistosäätiö and Jenny- ja Antti Wihuri foundation. The molecular work was supported with a grant from the Sir Mark Mitchell Foundation. A Geddes Award was provided by the Australian Museum to cover Weijola’s stay in Sydney while working on the AMS collections. We thank Gregory Schneider (UMMZ), Kathleen Imada (BPBM), Patrick Couper and Andrew Amey (Queensland Museum), Ross Sadlier (AMS), Paul Doughty (Western Australian Museum), and Chris Austin (LSUMZ) for providing tissue samples. Weijola is grateful to: Ross Sadlier, Patrick Couper, Andrew Amey, Paul Doughty, Ronald de Ruiter (Naturalis), Hielke Praagman (ZMA) and Daniel Klingberg Johansson (ZMUC) for providing access to their collections. Frank Tillack (MZB) provided information on specimens under his care. Thomas Ziegler kindly let us reproduce figures from his earlier works. We thank the people of Nai for their hospitality and help during Weijola’s stay on Mussau and Quetzal Dwyer for providing us with a photograph. David Abrahamsson and many others provided valuable assistance in the field. Sam Sweet and Daniel Bennett commented on a first draft of the manuscript and the former assisted greatly with the drawn figure. Lastly we thank Andre Koch and Fred Kraus who provided constructive and critical reviews on the submitted manuscript.