Three new species of Kynotus from the Central Highlands of Madagascar (Clitellata, Megadrili)

The earthworm fauna of Madagascar is scarcely known. A recently launched exploration of the soil fauna (“Global Change and Soil Macrofauna Diversity in Madagascar”) resulted in the discovery of six new earthworm species belonging to the Malagasy endemic family Kynotidae. The success of the collecting campaign carried out between 2008 and 2011 inspired a new exploration of the earthworm fauna across the Central Highland Region of the island in the spring of 2015. During this expedition, two new species of Kynotus, K. ankisiranus sp. nov. and K. voimmanus sp. nov., were discovered. Barcoding of the recently collected species of Kynotus revealed that the unpigmented worms referred previously to K. alaotranus Michaelsen, 1897 also represented a new, still undescribed species, K. blancharti sp. nov.


Introduction
Madagascar, with its territory of 592,800 km 2 , is the fourth largest island in the world and, due to its tectonic history and environmental variability, is listed among the 25 megadiverse regions of the earth (Myres et al. 2000).This is why the island's fauna has always been the focus of research (Goodman & Benstead 2003).Interestingly though, data on the earthworm fauna of Madagascar is quite scarce; since the description of the first earthworm species Geophagus darwini (=Kynotus darwini) by Keller (1887) just a couple of papers dealing with the earthworm fauna of Madagascar (e.g., Michaelsen 1891Michaelsen , 1897Michaelsen , 1907Michaelsen , 1931;;Rosa 1892) have been published.After the last paper by Michaelsen (1931), no new taxonomic works on the earthworm fauna of the island were published until a new project, entitled "Changement global et diversité de la macrofaune du sol à Madagascar" (Global Change and Soil Macrofauna Diversity in Madagascar), was launched in 2008.During this project, a comprehensive collecting campaign resulted in the discovery of several rare, as well as new, earthworm species (Razafindrakoto et al. 2010(Razafindrakoto et al. , 2011(Razafindrakoto et al. , 2016;;Csuzdi et al. 2012).
These results inspired new collecting activities carried out by the latter authors, which resulted in the discovery of two further species new to science, belonging to the endemic family Kynotidae.Barcoding of the recently collected material of Kynotus revealed that the unpigmented specimens, previously relegated to the red-pigmented K. alaotranus Michaelsen, 1907(Csuzdi et al. 2012), also represent a new species.Descriptions of the three new species of Kynotus, together with their barcodes, are herewith provided.

Study area
The new collecting sites are situated in the Central Highlands region of the island (Fig. 1).

Methods
Earthworms were collected primarily by using the diluted formaldehyde method (Raw 1959) supplemented by digging and hand-sorting.The material collected was killed in 75% ethanol and fixed in 4% formaldehyde solution.Parallel material from each morpho-species was conserved in 96% ethanol for DNA studies.

Deposition
The material collected is deposited in the Hungarian Natural History Museum (HNHM) and in the collection of Chonbuk National University, Korea (CHBNU).

Molecular methods
A small piece of the postclitellate body wall was cut out from the paratype specimens for barcoding, as indicated in Table 1, and processed according to the methodology described in Szederjesi & Csuzdi (2015).Additional COI sequences of Kynotus were acquired from the BOLD database (Table 1).COI sequences were aligned with ClustalW (Thompson et al. 1994), using the default settings.The final dataset was 671 bp long and contained no internal gaps.Maximum Likelihood analysis was carried out with Mega v. 6.06 (Tamura et al. 2013) using the best fitting substitution model GTR G + I and 1000 bootstrap replicates.Bayesian inference was performed with BEAST v. 1.8.2 software (Drummond et al. 2012) with the GTR G + I substitution model selected by MEGA using the Akaike Information Criterion.BEAST was run for 10 million generations, saving trees at every 1000 th generation.The first 2000 trees were discarded "burn in" in TreAnnotator v. 1.8.2.The resulting tree was visualized with Fig Tree v. 1.4.2 (Rambaut 2014).Inter-and intraspecific genetic distances were calculated with MEGA using the K2P substitution model.
Besides the lack of red pigments, the penial setae are also somewhat different.Instead of being lanceolate as in alaotranus, in the new species the tip is flanked with slightly dentate rims making the seta more spoon-shaped.K. blancharti sp.nov. is clearly different from all the examined alaotranus group species by its COI barcode as well (Table 3, Fig. 5).

Etymology
The species epithet refers to the type locality Voimma Community Park.

Remarks
This new species, with biannulate segments between 4 and 10 belongs to the alaotranus group (Table 2).In the alaotranus group there are only two other species possessing a single pair of genital setal glands: K. rosae Cognetti, 1906 andK. minutus Csuzdi, Razafindrakoto &Blanchart, 2012.K. voimmanus sp.nov.differs from K. rosae by its smaller number of spermathecal segments (13/14, 14/15, 15/16, 16/17 vs 13/14, 14/15, 15/16) and also in the structure of spermathecae (K.rosae possesses oval spermathecae with very short ducts, K. voimmanus sp.nov.possesses oval spermathecae with thin and very long ducts, 2-3 times longer that the ampoule).The new species differs from K. minutus in its size and Fig. 5. Bayesian inference tree of some species of Kynotus Michaelsen, 1891 using COI sequences.Numbers above branches indicate Bayesian posterior probabilities, below branches ML bootstrap supports.Values lower than 80% are not shown.coloration, and furthermore in the position of the clitellum (18-26 vs 18-½ 28), as well as the shape of the spermathecae (short vs long spermathecal duct).K. voimmanus sp.nov. is clearly different from all the examined alaotranus group species by its COI barcode as well (Table 3, Fig. 5).

Discussion
Before launching the project "Changement global et diversité de la macrofaune du sol à Madagascar" (Global Change and Soil Macrofauna Diversity in Madagascar) in 2008, only 13 species were known from the Malagasy endemic earthworm family Kynotidae.Our activity in the recent years has resulted in the description of a further 9 Kynotus species new to science (Razafindrakito et al. 2011;Csuzdi et al. 2012;Razafindrakoto et al. 2017), increasing the number of valid species in the family to 22.It is worth noting that, due to difficulties accessing the rural sites in the rainy season (which is the most suitable for earthworm collections), larger regions of the country, especially in the western and southern part of the island, still remain unexplored.It is also important to highlight that several new species were discovered in highly disturbed habitats such as around Sambaina (K.blancharti sp.nov. in secondary bushland, with scattered Eucalyptus L'Hér.) or Antsirabe (K.parvus Csuzdi, Razafindrakoto & Blanchart, 2012 in secondary Aristida L. grassland).This indicates that not only the very few pristine regions should be sampled, but also disturbed areas, where native, still undescribed, species occur.
Our barcoding results clearly separated all the newly described species (Fig. 5), supporting the conclusion that this method is quite powerful in recognizing earthworm species, as demonstrated recently by Decaëns et al. (2016), Chang & James (2011) and Chang et al. (2009).The COI tree revealed that the alaotranus group's species (except K. michaelseni) form the second largest clade and show a clear south-north geographical distribution pattern.The most basal species of this clade, K. ankisiranus sp.nov., possesses the southernmost distribution and the terminal species pair K. voimmanus sp.nov.-K.alaotranus is distributed in the north (Figs 1, 5).However, it seems that COI alone is not suitable for inferring the phylogenetic relationships inside the genus.All deeper branches revealed showed no statistical support.

Table 2 .
Distinguishing characters in the K. alaotranus species group.

Table 3 .
Average interspecific (below the diagonal) and intra specific (bold, in the diagonal) K2P genetic distances of the species of Kynotus Michaelsen, 1891 analysed.