New Polypheretima and Pithemera (Oligochaeta: Megascolecidae) species from the Mt. Malindang Range, Mindanao Island, Philippines

We describe four new pheretimoid earthworm species, one in Polypheretima and three in Pithemera, from Mt. Malindang, Misamis Occidental Province, Mindanao Island, Philippines, and provide diagrams of external morphology and internal anatomy. Polypheretima mindanaoensis sp. nov. belongs to the Po. elongata species group, characterised by having genital markings on xix and successive segments and pairs of spermathecal batteries in vi and/or vii. It differs from the other members of the Po. elongata species group in having no copulatory bursae. This species shows individual variation in the number of spermathecae in each battery. Individuals that lack spermathecae are presumed to reproduce parthenogenetically. Pithemera malindangensis sp. nov. and Pi. duminagati sp. nov. belong to the Pi. bicincta species group, characterised by having the first spermathecal pores in 4/5. These two species differ in size and in the distance between male pores. Pithemera donvictorianoi sp. nov. belongs to the Pi. pacifica species group, characterised by having the first spermathecal pores in 5/6. This is the only member of this species group so far reported from the Philippines, and this is the only Philippine Pithemera species whose clitellum covers two rather than two and a half segments. Current studies show that worldwide, the Philippines has the highest diversity for Pithemera, with 13 species, followed by Papua New Guinea and the Pacific Islands, each area with six species. Indonesia has the highest diversity for Polypheretima, with 18 species, followed by Vietnam with 13 species, and then Papua New Guinea and the Philippines, each area with eight species. These findings indicate a high degree of endemicity within these areas, suggesting that many species remain to be detected in the Philippines. http://zoobank.org/urn:lsid:zoobank.org:pub:2198709C-F5E1-4A0D-A185-CA506D206171


Introduction
Mt. Malindang Range Natural Park (MMRNP) comprises a complex of inactive volcanoes located at the foot of the Zamboanga Peninsula, which extends westward from central Mindanao Island, Philippines. Comprising an area of 533,000 km 2 , the Park ranges in altitude from 600 m above sea level (a.s.l.) in the lowlands to 2404 m a. s.l. at the top of the highest peak, Mt. Malindang (8. 2166°N, 123.6333°E). About 330,000 km 2 remain covered with relatively undisturbed primary forest, while the rest

Site description
Samples were collected from 9 to 15 October 2003 in primary and disturbed forests at different elevations in Barangay [= Precinct] Lake Duminagat in the municipality of Don Victoriano, Barangay Small Potongan in the municipality of Concepcion, and Barangays Sibucal and Toliyok in Oroquieta City ( Figure 1B). All are in Misamis Occidental Province. The primary forest had remained largely undisturbed by human activities. The vegetation was dense and lush; trees were stout and thickly covered with moss, ferns and lichens. The ground was also thickly covered with moss, roots and leaf litter. Trees in the disturbed forest, regrown after deforestation by humans, were dominated by dipterocarps, tended to be more closely spaced than in the primary forest and tended to have more undergrowth (saplings, shrubs and tree ferns). The ground was also covered with thick leaf litter, roots, bryophytes and lichens. The terrain in the forested areas was very rugged, with steep grades and many cliff faces, making access very difficult. Surrounded by humid primary forest in Barangay Lake Duminagat is a crater lake about 60 km 2 in area called Lake Duminagat. The geographical coordinates of collecting sites were determined by global positioning system (GPS; Magellan Map410); elevations were determined by GPS if a satellite signal was detectable, or with an altimeter if not. The map datum used in the GPS readings was Luzon.

Sampling
Sampling methods and the locations of sampling sites were previously reported in Aspe (2006) and Aspe et al. (2009). A summary is as follows: in each barangay, six scattered plots 20 m × 20 m in extent were established, with an average distance of 75 m between plots. On each plot, 10 quadrats (0.5 m × 0.5 m square × 0.3 m deep) in randomly selected spots were dug and searched for earthworms. The earthworms collected were preliminarily sorted to species and counted to assess relative abundance. Additional haphazard sampling was done outside the plots to further assess species' distributions across all sites. Tree bark, ferns, mosses, vines and the insides of rotten logs were also checked for earthworms. Earthworms collected were cleaned in tap water, killed in 10% ethanol, and placed in Saranex sealable plastic bags filled with a volume of 10% formalin that was at least three times the total volume of the earthworms. After 2 days, the formalin was replaced with 80% ethanol for long-term preservation.

Descriptions
The descriptions given below are based on the terminological conventions of Easton (1979), and the classification is that of Sims and Easton (1972). Descriptions of body colour are based on living specimens. Body dimensions refer to fixed material. The degree of separation between pores is expressed as a proportion of the circumference of the worm; for example, 'male pores 0.23 circumference apart ventrally' means the distance between the pores is 0.23 times the circumference of the worm at that point, with the circumference calculated as π times segment diameter. While many character states are shared among species within genera, generic characters of the three species in Pithemera are repeated to aid in identification. All descriptions are based on external examination and dorsal dissection under a stereomicroscope. Illustrations were prepared with Adobe Illustrator v. CS5. Holotypes and some paratypes are deposited in the Annelid Collection of the National Museum of the Philippines (NMA), Manila. Other paratypes are deposited in the Annelid Collection of the Zoological Reference Collection (ZRC.ANN) of the Raffles Museum of Biodiversity Research, Department of Biological Sciences, National University of Singapore, Singapore.

Results
We describe four new species from Mt. Malindang, Mindanao Island: Polypheretima mindanaoensis sp. nov., Pithemera malindangensis sp. nov., Pi. duminagati sp. nov. and Pithemera donvictorianoi sp. nov. Figure 1B shows the location in Mt. Malindang where Polypheretima mindanaoensis and species of Pithemera were collected. Table 1 shows the frequency, site density and relative abundance of the four species at the five collecting sites on Mt. Malindang. The numbers of Pheretima individuals (Aspe and James 2014) are also included to indicate the overall relative abundance and frequency of Polypheretima and Pithemera species in the earthworm community. Among species in the latter two genera, Pithemera malindangensis sp. nov. was the most abundant, with a relative abundance of 11.7%, while Pithemera Table 1. Density and frequency of earthworm species found at collecting sites on Mt. Malindang (modified from Aspe et al. 2009, tab. 2). Data are given for only five of nine sites, as no individuals were found at two of the sites (agricultural areas and grasslands in Barangays Small Potongan and Toliyok). + indicates individuals were also collected outside sampling plots. The density value for each site is individuals collected per 4.5 m 3 of soil examined on plots. * Aspe and James (2014).  Note: asl = above sea level.
duminagati sp. nov. showed the highest frequency of occurrence (0.6), though it was only collected through haphazard sampling outside the plots. The sites with the highest species diversity were in disturbed forest in Barangays Lake Duminagat and Sibucal (20 and 17 species, respectively). The sites with highest species diversity and abundance were all above 900 m in elevation. Polypheretima mindanaoensis sp. nov. and the three Pithemera species were not detected in Barangay Small Potongan, and only one individual of Pithemera donvictorianoi sp. nov. was collected in Barangay Toliyok at a lower elevation.

Taxonomy
Family MEGASCOLECIDAE Rosa, 1891 Genus Polypheretima Michaelsen, 1934 Type species. Perichaeta stelleri Michaelsen, 1892 Generic diagnosis Body cylindrical; setal arrangement perichaetine; annular clitellum covering segments xiv-xvi; pair of male pores in xviii on circular porophores which may be within copulatory bursae; ventral genital markings present or absent; oesophageal gizzard in viii; intestine begins in xv or xvi; nephridia on spermathecal ducts lacking; caeca lacking; male sexual system usually holandric, with testes and funnels enclosed in paired sacs in x and xi; seminal vesicles in xi and xii; spermathecal pores small, spermathecal diverticula simple and usually ectal in origin; prostates racemose; copulatory bursae may or may not be present; ovaries free in xiii; oviducts lead to single or closely paired opening (Easton 1979

Description
White, clitellum pinkish-grey. Body stout, adult length 90-118 mm; diameter 5.1 mm at x, 7 mm at xx; 140-141 segments (n = four adults); body cylindrical in crosssection, tail narrowing gradually to sharp point. First dorsal pore at 12/13; spermathecal pores lacking or inconspicuous; female pore single in xiv, male pores on paired low papillae on xviii, 0.23 circumference apart, 10 setae between openings. Clitellum annular, from xiv to xvi. Setae pointed posteriorly, unevenly distributed around segmental equators; 41-53 setae on vii, 44-46 setae on xx; dorsal and ventral setal gaps lacking. Genital markings widely paired on xix to xxv and/or xxvi, between the sixth and seventh setal lines. Septa 4/5/6/7/8 muscular, 8/9 absent, 9/10 present around dorsal vessel and hearts; 10/11 to 13/14 thickly muscular. Dense tufts of nephridia on anterior faces of 5/6 and 6/7; nephridia of intestinal segments located at septum-body wall junction, mainly on body wall near anterior and posterior faces of septa. Oesophageal gizzard large, extending from viii-x; oesophagus with lamellar inner surface extend from xi-xiii;  intestinal origin in xv, caeca lacking; typhlosole a simple fold of about one sixth lumen diameter originating in xvi; intestinal wall without longitudinal blood vessels but with two pairs of vertical vessels per segment.
Hearts in x to xii, oesophageal. Commissural vessels in vi, vii and ix, lateral; lacking in viii. Supra-oesophageal vessel extending from x to xii; extra-oesophageal vessels joining ventral oesophageal wall in x, receiving efferent parieto-oesophageal vessels in xiv.
Ovaries and funnels free in xiii, spermathecae lacking in two adults, one adult individual with five spermathecae closely lining up on the left side of vi, another adult individual with three spermathecae closely lining up on the left side and five spermathecae closely lining up on the right in vi and two spermathecae on left and two on right in vii; spermathecae small, ampulla pyriform, spermathecal duct short, slender; diverticulum stalk long and slender, attached ectally to duct, with one kink, terminating in short, sausage-shaped receptacle; no nephridia on spermathecal ducts. Male sexual system holandric, testes and funnels enclosed in paired ventral sacs in x and xi; seminal vesicles in xi and xii; pseudovesicles in xiii; vasa deferentia slender, free from body wall en route to ental end of prostatic ducts; prostates in xvi to xxi, each prostate racemose, trilobed but compact; copulatory bursae lacking.

Remarks
Polypheretima mindanaoensis sp. nov., the first member of Polypheretima reported from Mindanao Island, belongs to the Po. elongata Perrier, 1872 species group of Easton (1979), characterised by having a pair of genital markings in xix, successive segments in line with the male pores, paired batteries of up to 28 spermathecae in vi and/or vii, and shallow copulatory bursae with no stalked glands. Easton (1979) included five species in the group: Po. elongata; Po. everetti; Po. kinabaluensis Beddard and Fedarb, 1895;Po. phacellotheca Michaelsen, 1899;and Po. stelleri Michaelsen, 1892. Polypheretima mindanaoensis markedly differs from Po. elongata and Po. everetti in size (355 × 4 in Po. elongata; 300 × 12 in Po. everetti), in the number of segments covered by the genital markings (extending from xix to xxii in Po. elongata; xix to xxi in Po. everetti) and in the number of setae on vii (usually 80-130 in Po. elongata; 130 in Po. everetti) ( Table 2). Polypheretima mindanaoensis (white) differs in colour from Po. everetti and Po. kinabaluensis (reddish-purple and red, respectively). Those individuals of Polypheretima mindanaoensis that have spermathecae have fewer spermathecae in each battery than Po. kinabaluensis (6-12 in 5/6/7), Po. phacellotheca (9-12 in 5/6) or Po. stelleri (up to 28 in 5/6/7), and individuals have fewer setae (41-53) on vii than Po. phacellotheca (80) Easton (1979), characterised by having pairs of spermathecae in 5/6-8/9 or only in 6/7 or 7/8, while Po. fruticosa is closely related to Po. voeltzkowi Michaelsen, 1907, characterised by having a pair of spermathecae in only in 5/6. The new species also differs from the other species in the number of setae between male pores, in the genital marking pattern and in the size and segmental position of the prostate glands (Table 2). Spermathecae function to receive and store sperm released by the male pores of the partner during copulation; loss of spermathecae usually means a loss of male function. Loss of spermathecae, reduction of testes and lack of spermatozoal iridescence in the sperm funnels in earthworms are indicative of reproduction by parthenogenesis (Gates 1972), in which the egg develops into a new individual without being fertilised by sperm. In the case of Po. elongata, which has been introduced into many parts of the world (see Table 4), Easton (1979) observed that athecate individuals are especially numerous in introduced populations, presumably reproducing parthenogenetically.
In one specimen of Po. mindanaoensis sp. nov., the ventral nerve cord has a solid dark core, perhaps related to parasitism. We observed several nematodes near an empty nerve cord swelling in xvii.

Occurrence
Polypheretima mindanaoensis sp. nov. was found at two of five collecting sites. In all, eight individuals were collected in disturbed forests in Barangays Lake Duminagat and Sibucal at elevations of 902-1662 m a.s.l. The soil it inhabited was covered with thick leaf litter and roots, bryophytes and lichens. We did not observe it on rotten logs.
Genus Pithemera Sims and Easton, 1972 Type species. Perichaeta bicincta Perrier, 1875 Generic diagnosis Body cylindrical; setae numerous, regularly arranged around each segment; clitellum annular, covering two or two and a half segments from xiv to xv, and/or half of xvi; spermathecal pores small, three to five pairs from 4/5 to 8/9; female pore single or paired in xiv; genital markings present or absent; oesophageal bursae absent; intestinal caeca originating in xxii (in Pheretima, caeca originating in xxvii); spermathecal ducts without nephridia (as in Polypheretima); male system holandric or metandric; prostate glands racemose; copulatory bursae lacking (Sims and Easton 1972). Hong and James (2008a) observed that Pithemera species are generally lighter in pigmentation and smaller in size than Pheretima species. Pithemera malindangensis sp. nov.

Etymology
The species is named for Mt. Malindang.
Hearts in x-xii, oesophageal. Commissural vessels in vi, vii and ix, lateral; lacking in viii. Supra-oesophageal vessel extending from x to xii; extra oesophageal vessels joining ventral oesophageal wall in x, receiving efferent parieto-oesophageal vessels in xiv.
Ovaries and funnels free in xiii. Spermathecae five pairs in v to ix; duct short, slender; small, spherical to ovate ampulla greater than duct length; diverticulum stalk short, attached ectally to duct, terminating in short, sausage-shaped receptacle. Male sexual system holandric; testes and funnels enclosed in paired ventral sacs in x and xi; seminal vesicles in xi and xii; vasa deferentia slender, free from body wall, those on each side separate until xiv, joining ental end of prostatic ducts; each prostate racemose, broad, multilobed, in xvi-xix; ectal half of duct expands to form muscular spindle; ducts in hairpin loop. Transverse muscle bands present above body wall at 16/17 and 18/19, the latter much larger. Copulatory bursae lacking.

Remarks
Pithemera malindangensis sp. nov. belongs to the Pi. bicincta (Perrier, 1875) species group of Sims and Easton (1972), which initially comprised two species, Pi. bicincta and Pi. violacea Beddard, 1895. Michaelsen (1910, Ohfuchi (1957) and Shen and Tsai (2002), however, considered Pi. violacea to be a junior synonym of Pi. bicincta. Pithemera bicincta, first collected from Mindoro Island, has the first spermathecal pores in 4/5, and the intestinal caeca are paired and positioned laterally. Pithemera malindangensis is similar to Pi. bicincta in the arrangement and number of spermathecae and septa, the number and locations of hearts, and the length of the caeca, but the former is larger, lacks genital markings, and has the intestinal origin in xvi rather than xv. Other members of the bicincta species group reported from the Philippines (all from Luzon Island except for Pi. duminagati sp. nov. described below) include Pi. rotunda and Pi. philippinensis ); Pi. duhuani, Pi. fragumae, Pi. ifugaoensis and Pi. triangulata (Hong and James 2008a); and Pi. glandis, Pi. fusiformis and Pi. levii (Hong and James 2011a). Pithemera malindangensis sp. nov. and Pi. duminagati sp. nov. are similar to most or all other members of the bicincta group in the origin of the gizzard, number of hearts, and length and location of the caeca, but differ from the others in having the intestinal origin in xvi rather than xv. The Malindang species are also white and lack genital markings. Pithemera malindangensis sp. nov. is larger than Pi. duminagati sp. nov., the male pores are more distant and it has only two pairs of dense tufts of nephridia (on the anterior faces of 5/6/7), whereas the latter has three pairs (on 5/6/7/8; Table 3).
Occurrence Pithemera malindangensis sp. nov. was the most abundant among the four species, comprising 11.7% of all earthworms collected. It was common in primary and disturbed forests in Barangay Lake Duminagat at elevations of 1479-2027 m a.s.l. The soil it inhabited was covered with thick leaf litter and roots, bryophytes and lichens. We did not observe it on rotten logs. Pithemera duminagati sp. nov.

Etymology
The species is named for Barangay Lake Duminagat, the type locality. nov.
Hearts in x-xii, oesophageal; commissural vessels in vi, vii and ix, lateral; lacking in viii; supra-oesophageal vessel extends from x to xii; extra oesophageal vessels joining ventral oesophageal wall in x, receiving efferent parieto-oesophageal vessels in xiv.
Ovaries and funnels free in xiii; ovisacs in xiv; spermathecae five pairs from v to ix; duct short, slender; small, ovate ampulla; diverticulum stalk short, attached ectally to duct, terminating in short, pyriform receptacle; receptacle a small sphere lacking spermatozoal iridescence. Male sexual system holandric; testes and funnels enclosed in paired ventral sacs in x and xi; seminal vesicles in xi and xii; vasa deferentia slender, free from body wall, joining ental end of prostatic ducts; each prostate racemose, broad, 3-or 4-lobed, present from xvi to xxi; ectal half of duct expands to form muscular spindle; ducts in hairpin loop. Ental portion of duct with two lumens. Copulatory bursae lacking.

Remarks
Pithemera duminagati sp. nov. belongs to the Pi. bicincta group of Sims and Easton (1972). Although Pi. duminagati sp. nov. is similar to Pi. bicincta in some characters, it differs from the latter in size, in lacking genital markings, in the distance between male pores, in the relative size of the prostates and in the origin of the intestine (Table 3). Pithemera duminagati sp. nov. is similar to Pi. rotunda , another member of the bicincta group, in size and in being white, in septal arrangement, in intestinal origin and in the length of caeca, but Pi. duminagati sp. nov. has fewer setae between the male pores, lacks genital markings, and has proportionally larger prostate glands. Among the three Pithemera species from Mt. Malindang, Pi. duminagati sp. nov. is more similar to Pi. malindangensis sp. nov. in the extent of the clitellum, number of spermathecae and septal arrangement. Like Pi. donvictorianoi sp. nov., Pi. duminagati sp. nov. is smaller in body size than Pi. malindangensis sp. nov. and has three pairs of dense tufts of nephridia on 5/6/7/8, whereas the latter has only two pairs on 5/6/7.
Occurrence Pithemera duminagati sp. nov. was found outside the sampling plots at three of five sites: in primary and disturbed forest in Barangay Lake Duminagat and in disturbed forest in Barangay Sibucal, at elevations of 902-2027 m a.s.l. The soil it inhabited was covered with thick leaf litter and roots, bryophytes and lichens. We did not observe it on rotten logs.

Etymology
The species is named for the municipality of Don Victoriano, the type locality.
Hearts in x-xii, oesophageal; commissural vessel in vi, vii and ix, lateral; viii extending to gizzard; supra-oesophageal vessel extending from x to xii; extra-oesophageal vessels joining ventral oesophageal wall in x, receiving efferent parietooesophageal vessels in xiv.
Ovaries and funnels free in xiii; ovisacs lacking; spermathecae four pairs in vi to ix; duct short, slender; ampulla small, narrow ovate; diverticulum stalk short, attached ectally to duct, terminating in short, ovate receptacle. Male sexual system holandric; testes and funnels enclosed in annular sacs in x and xi; with sacs enclosing hearts; seminal vesicles in xi-xii; vasa deferentia slender, free from body wall, joining ental end of prostatic ducts; prostates in xvi to xix; each prostate racemose, broad, 4-lobed, lobes deeply incised; ectal half of duct expands to form muscular spindle; ducts in hairpin loop. Ental portion of duct with three lumens. Copulatory bursae lacking.

Remarks
Pithemera donvictorianoi sp. nov. belongs to the Pi. pacifica group of Sims and Easton (1972) and is the only member of this species group so far reported from the Philippines. This group is characterised by having four spermathecae, with the first spermathecal pore in 5/6, in contrast to the Pi. bicincta group, members of which have the first pair of spermathecal pores in 4/5. Like the other Pithemera species on Malindang, it is white and lacks genital markings, and the caeca extend from xvii to xxi. Like Pi. duminagati sp. nov., it has smaller average body size than Pi. malindangensis sp. nov., and it has three pairs of dense tufts of nephridia, on 5/6/7/8, whereas Pi. malindangensis has only two pairs, on 5/6/7. Pithemera donvictorianoi differs from all other Philippine Pithemera species in that the clitellum extends from xiv through xv, rather than from xiv through half of xiv (Table 2).
Occurrence Pithemera donvictorianoi sp. nov. was rare, with only one individual collected inside a plot in disturbed forest in Barangay Toliyok, though we also detected it in disturbed forest in Barangay Lake Duminagat. The soil it inhabited was covered with thick leaf litter and roots, bryophytes and lichens. We did not observe it on rotten logs.

Discussion
Blakemore (2007) listed 14 valid Pithemera species distributed in East Asia and the Pacific region, described from 1938 to 2004. He also listed 38 valid Polypheretima species distributed from East Asia and the Pacific region to South America and Africa, described from 1872 to 1984. In addition, Nguyen et al. (2014Nguyen et al. ( , 2015 listed a total of 13 Polypheretima species from Vietnam, which includes four recently described species. Prior to 2004, only one Pithemera and three Polypheretima species were known from the Philippines: Pithemera bicincta from Mindoro Island; Polypheretima monticola from Benguet Province, Luzon Island; and Po. elongata and Po. everetti, both from Balabac Island, Palawan Province. From 2004 to present (including this study), 12 Pithemera and five Polypheretima species were added to the Philippine fauna, bringing the totals to 13 and eight, respectively. Table 4 shows the diversity of Polypheretima and Pithemera species reported from the various countries or regions where they occur. The Philippines has the highest diversity of Pithemera, with 13 species, followed by Papua New Guinea and the Pacific Islands, each area with six species. For Polypheretima, Indonesia has the highest diversity, with 18 species, followed by Vietnam with 13 species and then Papua New Guinea and the Philippines, each area with eight species. Among the species of Polypheretima, Po. elongata is the most widespread, having been reported from 10 regions around East Asia, the Pacific and Africa. This is followed by Po. taprobanae and Po. everetti, reported from six and four regions, respectively, around Asia, the Pacific, South America and Madagascar. The most widespread Pithemera species is Pi. bicincta, which occurs in six regions around East Asia and the Pacific. Among the species in the Philippines, Po. elongata, Table 4. Diversity of Polypheretima and Pithemera species around the world. Symbols indicate species with broad ranges: *Polypheretima elongata; + Polypheretima everetti; @ Polypheretima taprobanae; # Pithemera bicincta. These data are from Tsai et al. (2000); Blakemore (2003Blakemore ( , 2007; James (2004b); James et al. (2004); James (2008a, 2011a); Nguyen et al. (2014Nguyen et al. ( , 2015.

Country/region
Polypheretima Pithemera Po. everetti and Pi. bicincta are widely distributed, both locally and globally. Joshi et al. (1999) reported Po. elongata and Pi. bicincta at Ifugao, Luzon Island. The known ranges of the rest of the Pithemera and Polypheretima species are all restricted to the type localities, indicating a high degree of endemicity, both among local areas and islands in the Philippines, and in the Philippines as a whole. This pattern suggests that many species remain to be detected in the Philippines.
To facilitate the further study of earthworms at Mt. Malindang and on Mindanao Island, we provide the following key to the species that are from the Mt. Malindang Range. The key also includes the genus Pheretima and the exotic species Pontoscolex corethrurus. The most easily located external features are used at the beginning of the key. Until more is known about the taxonomy and distributions of earthworms on Mindanao and in the rest of the Philippines, however, this key should be used with caution in identifying earthworms from outside the Mt. Malindang Range.

Disclosure statement
No potential conflict of interest was reported by the author(s).