Five new cryptic freshwater gastropod species from New Caledonia (Caenogastropoda, Truncatelloidea, Tateidae).

During the course of a project aiming at the reconstruction of the colonization of the South Pacific islands by tateid gastropods based on molecular data we discovered five new species on New Caledonia belonging to the genera Hemistomia and Leiorhagium, respectively. We describe these species based on morphological, anatomical and genetic data. All five species are morphologically cryptic as they closely resemble or are even indistinguishable from known species stressing the importance of a comprehensive taxonomic approach integrating several methods. As a consequence of their small and fragmented geographic ranges and the rapidly progressing anthropogenic land cover changes on New Caledonia, all five species qualify as critically endangered according to the criteria of the IUCN.

Introduction

New Caledonia is famous for being a biodiversity hotspot harboring a high number of endemic species (Myers et al. 2000) including a radiation of small freshwater gastropods belonging to the family . This radiation is probably of Oligocene origin and comprises more than 50 species in seven genera (Haase and Bouchet 1998, Zielske and Haase 2015). Many of these species are extreme narrow-range endemics known from only few or single sites (Haase and Bouchet 1998), a pattern typical for in freshwaters worldwide (e.g. Giusti and Pezzoli 1980, Radoman 1983, Haase 1996, 2008, Ponder and Colgan 2002, Liu and Hershler 2005, Hershler et al. 2011, Delicado and Ramos 2012). In the frame of a project aiming at the reconstruction of the colonization of the South Pacific islands by tateids based on molecular data (Zielske and Haase 2014a, b, 2015, Zielske, Ponder and Haase in preparation) we visited New Caledonia in May 2012 in order to collect suitable material for sequencing. During this expedition we found five new species of the genera Crosse, 1872 and Haase & Bouchet, 1998, respectively (Figs 1, 2), which we describe herein based on morphological, anatomical and genetic data. All five species qualify as morphologically cryptic as they closely resemble or are even indistinguishable from known species (see Pfenninger and Schwenk 2007). The discovery of new cryptic species was predicted by Haase and Bouchet (1998), whose revision was based solely on morphology and anatomy. In general, cryptic species are common among different spring snail families of (e.g., Liu et al. 2003; Haase et al. 2007; Delicado and Ramos 2012; Collado et al. 2013).

Figure 1.

Localities of new species and samples used for morphometric comparisons. Inset shows position of New Caledonia in the Southwest Pacific. Arrows indicate type localities of species represented by more than one sample (see also Table 1).

Figure 2.

Holotypes. A sp. n. B sp. n. C sp. n. D sp. n. E sp. n. Scale bar = 1 mm.

Table 1.

Locality data of all samples and GenBank accession numbers of specimens represented in phylogeny (see also Fig. 1). The last three species represent the outgroup. Specimens are only distinguished in two cases where more than 1 sequence per sample was used. For museum catalog numbers of NeCa-sample voucher material see Zielske and Haase (2015). Paratypes of species described by Haase and Bouchet (1998) used in morphometric comparisons are accompanied by catalog numbers from the museum in Paris, because these have been assigned only recently.

Species, sample	Locality	Latitutde, longitude	COI	16S	IT2
Hemistomia andreae, NeCa 12_1 Hemistomia andreae, NeCa 12_2	Bouloupari, Ouaméni valley	21°49'46.9"S; 165°56'42.9"E	KJ490851 KJ490852	KJ490767 ---	KJ490691 ---
Hemistomia cockerelli, paratypes MNHN IM-2012-2694	Bouloupari, Ouaméni, prop. Debels	21°49'12.0"S; 166°56'36.0"E
Hemistomia cockerelli, NeCa 11	Bouloupari, Ouitchambo	21°48'16.8"S; 166°00'00.8"E
Hemistomia cockerelli, NeCa 17	Moindou, road toward barrage	21°39'52.8"S; 165°43"10.3"E	KJ490857	KJ490772	KJ490696
Hemistomia cockerelli, NeCa 21A	Farino, Sentier de la Cascade et des Sources	21°38'11.9"S; 165°46'36.6"E	KJ490863	---	KJ490702
Hemistomia cockerelli, NeCa 36	Sarraméa, track to “Trou d’Eau”	21°38'22.1"S; 165°51'37.5"E
Hemistomia cockerelli, NeCa 54	Hienghène, Tendo	20°42'54.7"S; 164°49'20.8"E
Hemistomia eclima, NeCa 19	Moindou, road toward barrage	21°39'58.4"S; 165°43'08.2"E	KJ490858	KJ490773	KJ490697
Hemistomia fabrorum, NeCa 1	Dumbéa, Koé, prop. Oesterlin	22°08'59.0"S; 166°29'10.6"E	KJ490829	KJ490749	KJ490670
Hemistomia fabrorum, NeCa 25B	Sarraméa, road side of RPN 5	21°34'15.7"S; 165°49'41.2"E	KJ490867	KJ490781	KJ490704
Hemistomia minor, NeCa 30	Moindou, road side SW Katrikoin	21°34'21.6"S; 165°41'02.5"E	KJ490872	KJ490786	KJ490709
Hemistomia nyo, NeCa 35	Bourail, Oua Oué	21°36'50.3"S; 165°35'31.5"E	KJ490880	KJ490791	KJ490716
Hemistomia oxychila, NeCa 43A	Poya, road side between Nétéa and Goipin	21°16'06.0"S; 165°14'32.0"E	KJ490893	KJ490804	KJ490726
Hemistomia rusticorum, NeCa 6A	Bouloupari, road side N Nassirah	21°48'08.0"S; 166°04'14.6"E	KJ490836	KJ490755	KJ490677
Hemistomia winstonefi, NeCa 3B	Mont Dore, Rue des Roseaux, prop. Solier	22°15'42.4"S; 166°34'08.7"E	KJ490834	KJ490753	KJ490675
Leiorhagium adioincola, NeCa 43B	Poya, side of road to Goipin	21°16'06.0"S; 165°14'32.0"E	KJ490895	KJ490806	KJ490728
Leiorhagium adioincola, NeCa 49	Poya, stream into Grotte d‘Adio	21°15'24.4"S; 165°14'46.4"E	KJ490901	KJ490812	KJ490734
Leiorhagium ajie, paratypes MNHN IM-2012-2688	Houailou, Néoua	21°24'00.0"S; 165°38'54.0"E
Leiorhagium aremuum, NeCa 33_1 Leiorhagium aremuum, NeCa 33_2	Moindou, Aremu valley	21°35'04.8"S; 165°39'07.5"E	KJ490878 KJ490879	KJ490789 KJ490790	KJ490714 KJ490715
Leiorhagium clandestinum, NeCa 30B	Moindou, road side SW Katrikoin	21°34'21.6"S; 165°41'02.5"E	KJ490874	---	KJ490711
Leiorhagium douii, paratypes MNHN IM-2012-2681	Poya, Grotte d’Adio	21°15'30.0"S; 165°14'30.0"E
Leiorhagium inplicatum, NeCa 9B	Bouloupari, road side of RP 4	21°44'30.9"S; 166°05'57.9"E	KJ490845	KJ490762	KJ490685
Leiorhagium kavuneva, paratypes MNHN IM-2012-2690	Sarraméa, prop. Bonnard	21°39'00.0"S; 165°50'48.0"E
Leiorhagium kavuneva, NeCa 15B	Bouloupari, Oua Tom	21°47'24.4"S; 165°54'51.2"E	KJ490855	KJ490770	KJ490694
Leiorhagium kavuneva, NeCa 27	Kouaoua, road side N Koh	21°30'52.2"S; 165°48'05.0"E	KJ490869	KJ490783	KJ490706
Leiorhagium kavuneva, NeCa 29	Kouaoua, road side N Koh	21°32'02.6"S; 165°49'27.2"E
Leiorhagium monachum, paratypes MNHN IM-2012-2679	Poya, Mt. Krapé	21°23'12.0"S; 165°14'30.0"E
Leiorhagium montfaouense, paratypes MNHN IM-2012-2684	Poya, Montfaoué	21°16'48.0"S; 165°17'42.0"E
Leiorhagium neteae, NeCa 44B	Poya, beginning of road into Vallée d’Adio	21°14'47.9"S; 165°15'45.0"E	KJ490897	KJ490808	KJ490730
Leiorhagium orokau, NeCa 42	Poya, near Nétéa	21°16'32.2"S; 165°12'17.6"E	KJ490891	KJ490802	KJ490724
Leiorhagium orokau, NeCa 57	Hienghène, Tendo	20°42'43.9"S; 164°47'47.5"E	KJ490912	KJ490823	KJ490744
Crosseana crosseana, NeCa 51	Koumac, seepage in N of town	20°32'32.2"S; 164°18'33.0"E	KJ490904	KJ490815	KJ490737
Crosseana melanosoma, NeCa 50	Voh, Boyen, overflow of reservoir	20°49'13.6"S; 164°36'56.4"E	KJ490902	KJ490813	KJ490735
Kanakyella gentilsiana, NeCa 58	Hienghène, Tendo	20°42'22.4"S; 164°47'20.0"E	KJ490914	KJ490825	KJ490746

Material and methods

Snails were fixed in 70% ethanol in the field, transferred to propylene glycol for shipping by courier, and returned to ethanol, this time 96%, after arrival in our lab. For measurements, up to 20 snails per sample were photographed under a Zeiss SteREO Discovery.V20 dissecting microscope with a Zeiss Axio Cam MR3. Five dimensions – shell height, shell width, aperture height, aperture width, body whorl width – were measured using the program AxioVision 40 V4.8. (Zeiss) and whorls counted to the nearest eighth (Kerney and Cameron 1979). Up to six shells were dissolved in diluted hydrochloric acid for dissections. Anatomies were photographed as well. These digital images served as template for drawings made on a graphical tablet. For scanning electron microscopy up to three shells, radulae and opercula were cleaned in 5% sodium hypochlorite. The cephalopodia of up to two males were dried using hexamethyldisilazane (Nation 1983). After sputter coating with gold objects were investigated in a Zeiss EVO LS10 Scanning Microscope.

Morphometric analyses of shell measurements including canonical variates analyses (CVA) maximizing the differentiation of groups in multivariate space, , assignment tests, and Hotelling’s T2-tests were conducted PageBreakin PAST 2.12 (Hammer et al. 2001). Sequential Bonferroni-correction was applied to multiple tests. These analyses also included samples of known, similar species the new ones could be mistaken for (Table 1). The selection of species used in comparisons was based on the phylogenetic analysis.

multivariate analyses of variance

Phylogenetic analyses were based on a selection of sequences generated by Zielske and Haase (2015), who analyzed fragments of the mitochondrial genes and 16S rRNA as well as the nuclear . For lab protocols see Zielske and Haase (2014a, 2015). We restricted the analysis to 3 specimens per species at most and used , , and as outgroups (Table 1). The alignment of 16S rRNA and ITS2 was generated using secondary structure information using RNAsalsa 0.8.1 (Stocsits et al. 2009) (for details see Zielske and Haase 2015) and checked for ambiguous and randomly similar sites in Aliscore 2.0 (Misof and Misof 2009). We defined seven partitions. PartitionFinder 1.1.0 (Lanfear et al. 2012) identified the following scheme and substitution models as optimal among all possible combinations of separate and merged partitions: COI 1st positions (TrNef+I), COI 2nd positions (F81), COI 3rd positions (TVM+I+Γ), 16S rRNA loops (TrN+ Γ), ITS2 loops (TrNef+I+Γ), joint stems of 16S rRNA and ITS2 (K80+I). With these settings, tree reconstructions were conducted in a framePageBreakPageBreakPageBreakwork using GARLI 2.01 (Zwickl 2006) with 500 replicates. Robustness was assessed by bootstrapping with 200 replicates.

cytochrome oxidase subunit I

internal transcribed spacer 2

maximum likelihood

Type and non-type material is deposited at the and at the .

Museum National d’Histoire Naturelle in Paris

Naturhistorisches Museum Wien

Results

Systematic descriptions

Diagnoses and descriptions of and and data used in our comparisons with the new species were provided by Haase and Bouchet (1998). Locality data include site number, district capital, site, coordinates, and date of collection. Shell measurements are given in Table 2 and not repeated in the descriptions.

Table 2.

Morphometry. Measurements in mm. Shell measures: AH; AW; BWW; SH; SW; W; statistics: CV; max; min; SD. First line of new species contains measurements of holotypes. Note that the holotype was only in case of included in the descriptive statistics. Numbers of whorls were only counted in the new species as this parameter was not used in the statistical analyses.

, aperture hight

, aperture width

, width of body whorl

, shell height

, shell width

, number of whorls

, coefficient of variation corrected for unequal sample sizes

, maximum

, minimum

, standard deviation

New species
	SH	SW	AH	AW	BWW	SH/SW	W
Hemistomia andreae sp. n. (N=20)	2.70	1.25	0.90	0.87	1.08	2.17	5.4
min	2.40	1.10	0.80	0.75	0.97	2.00	4.50
max	2.78	1.28	0.93	0.91	1.08	2.35	5.50
mean	2.60	1.18	0.85	0.82	1.02	2.20	5.14
median	2.60	1.17	0.85	0.82	1.01	2.23	5.25
SD	0.11	0.05	0.04	0.04	0.03	0.11	0.28
CV	4.40	3.93	4.23	4.54	2.77	4.94	5.49
Leiorhagium adioincola sp. n. NeCa 49 (N=20)	2.29	1.24	0.88	0.87	1.09	1.84	4.50
min	2.10	1.16	0.83	0.83	1.04	1.71	4.13
max	2.42	1.31	0.96	0.96	1.15	1.90	4.75
mean	2.25	1.25	0.88	0.89	1.10	1.80	4.36
median	2.24	1.24	0.88	0.89	1.10	1.80	4.25
SD	0.09	0.04	0.04	0.03	0.03	0.05	0.18
CV	4.21	2.96	4.15	3.92	2.94	2.91	4.24
Leiorhagium aremuum sp. n. (N=20)	2.19	1.35	0.97	0.91	1.16	1.62	4.25
min	2.03	1.29	0.87	0.86	1.10	1.53	3.75
max	2.43	1.46	1.03	1.00	1.25	1.69	4.25
mean	2.19	1.35	0.94	0.92	1.16	1.62	4.03
median	2.15	1.35	0.93	0.92	1.17	1.62	4.00
SD	0.11	0.05	0.04	0.04	0.04	0.04	0.15
CV	4.92	4.06	4.76	4.54	3.77	2.71	3.78
Leiorhagium clandestinum sp. n. (N=4)	2.49	1.32	0.94	0.95	1.16	1.91	4.50
min	2.23	1.26	0.89	0.88	1.07	1.77	4.25
max	2.49	1.32	0.94	0.95	1.16	1.91	4.50
mean	2.38	1.28	0.91	0.92	1.10	1.86	4.41
median	2.41	1.27	0.90	0.93	1.09	1.89	4.44
SD	0.11	0.03	0.02	0.03	0.04	0.06	0.12
CV	4.83	2.30	2.68	3.44	4.00	3.52	2.89
Leiorhagium neteae n. sp. (N=18)	2.07	1.12	0.75	0.77	0.91	1.84	4.50
min	1.85	0.97	0.65	0.70	0.82	1.76	4.25
max	2.23	1.17	0.79	0.80	0.95	2.01	5.00
mean	2.05	1.09	0.73	0.75	0.88	1.88	4.46
median	2.04	1.10	0.73	0.75	0.87	1.88	4.38
SD	0.12	0.05	0.03	0.03	0.03	0.07	0.19
CV	6.05	4.82	4.71	4.69	3.99	3.72	4.25
Material for comparisons
	SH	SW	AH	AW	BWW	SH/SW
Hemistomia cockerelli Types (N=20)
min	2.58	1.18	0.88	0.83	1.03	2.05
max	3.21	1.39	1.03	0.97	1.16	2.40
mean	2.79	1.27	0.94	0.91	1.09	2.19
median	2.74	1.25	0.93	0.90	1.09	2.18
SD	0.17	0.06	0.04	0.04	0.05	0.09
CV	6.20	4.91	4.31	4.52	4.23	4.36
Hemistomia cockerelli NeCa11 (N=20)
min	2.20	1.06	0.77	0.73	0.94	1.93
max	2.48	1.25	0.87	0.91	1.04	2.28
mean	2.33	1.13	0.81	0.81	0.97	2.06
median	2.32	1.12	0.80	0.80	0.96	2.03
SD	0.08	0.04	0.03	0.04	0.02	0.10
CV	3.49	3.70	3.36	4.60	2.36	4.75
Hemistomia cockerelli NeCa17 (N=20)
min	2.35	1.16	0.83	0.83	1.04	1.96
max	2.62	1.28	0.92	0.93	1.14	2.19
mean	2.50	1.21	0.87	0.87	1.08	2.07
median	2.51	1.21	0.87	0.88	1.08	2.07
SD	0.07	0.04	0.03	0.03	0.02	0.07
CV	2.90	3.20	3.16	3.25	2.32	3.43
Hemistomia cockerelli NeCa21A (N=8)
min	2.26	1.09	0.74	0.77	0.96	2.03
max	2.74	1.23	0.89	0.87	1.08	2.38
mean	2.49	1.17	0.84	0.83	1.03	2.12
median	2.49	1.17	0.85	0.83	1.05	2.09
SD	0.14	0.05	0.04	0.03	0.04	0.11
CV	5.87	4.33	5.41	3.89	3.66	5.50
Hemistomia cockerelli NeCa36 (N=13)
min	2.32	1.14	0.79	0.82	1.03	1.97
max	2.64	1.23	0.91	0.91	1.12	2.14
mean	2.43	1.18	0.85	0.85	1.06	2.05
median	2.42	1.19	0.86	0.85	1.06	2.04
SD	0.10	0.03	0.03	0.03	0.03	0.05
CV	4.25	2.42	3.79	3.22	2.64	2.64
Hemistomia cockerelli NeCa54 (N=20)
min	2.28	1.16	0.78	0.82	1.04	1.86
max	2.63	1.31	0.96	0.93	1.14	2.14
mean	2.47	1.23	0.87	0.88	1.09	2.00
median	2.47	1.23	0.86	0.87	1.10	2.02
SD	0.10	0.04	0.04	0.03	0.03	0.07
CV	4.18	3.08	4.18	3.28	2.62	3.62
Hemistomia nyo NeCa35 (N=7)
min	2.43	1.25	0.88	0.89	1.09	1.93
max	2.75	1.34	0.96	0.96	1.15	2.08
mean	2.62	1.30	0.92	0.92	1.12	2.01
median	2.69	1.30	0.91	0.91	1.11	2.03
SD	0.12	0.04	0.03	0.03	0.02	0.06
CV	4.80	2.84	3.30	3.10	2.00	3.00
Leiorhagium ajie Types (N=6)
min	2.35	1.31	0.93	0.94	1.12	1.61
max	2.74	1.62	1.10	1.06	1.34	1.80
mean	2.50	1.46	1.01	1.00	1.25	1.72
median	2.43	1.46	1.01	1.00	1.27	1.70
SD	0.16	0.12	0.07	0.05	0.08	0.07
CV	6.50	8.31	6.95	4.88	6.32	4.12
Leiorhagium douii Types (N=20)
min	1.87	0.98	0.68	0.68	0.86	1.84
max	2.50	1.16	0.84	0.79	0.97	2.16
mean	2.06	1.05	0.73	0.71	0.91	1.96
median	2.02	1.06	0.72	0.71	0.91	1.95
SD	0.14	0.04	0.04	0.02	0.03	0.08
CV	7.04	4.02	5.11	3.51	3.21	4.23
Leiorhagium kavuneva Types (N=20)
min	2.17	1.17	0.78	0.82	1.02	1.77
max	2.42	1.32	0.94	0.93	1.13	1.93
mean	2.31	1.26	0.88	0.88	1.07	1.84
median	2.33	1.25	0.89	0.88	1.07	1.85
SD	0.07	0.04	0.04	0.03	0.03	0.05
CV	3.24	3.16	4.52	3.15	2.87	2.58
Leiorhagium kavuneva NeCa15B (N=20)
min	2.20	1.21	0.84	0.88	1.07	1.76
max	2.46	1.31	0.94	0.98	1.20	1.97
mean	2.34	1.27	0.90	0.92	1.12	1.84
median	2.35	1.28	0.91	0.92	1.12	1.83
SD	0.07	0.03	0.03	0.03	0.03	0.06
CV	3.14	2.30	3.31	3.00	2.54	3.14
Leiorhagium kavuneva NeCa29 (N=20)
min	2.17	1.20	0.85	0.85	1.06	1.76
max	2.54	1.36	1.00	0.99	1.17	1.97
mean	2.35	1.28	0.91	0.93	1.12	1.83
median	2.34	1.27	0.90	0.93	1.13	1.82
SD	0.10	0.04	0.04	0.04	0.03	0.05
CV	4.27	3.12	4.50	3.93	2.91	2.62
Leiorhagium monachum Types (N=3)
min	2.07	1.04	0.72	0.69	0.88	1.88
max	2.18	1.10	0.82	0.78	0.97	2.00
mean	2.11	1.08	0.76	0.74	0.92	1.96
median	2.07	1.09	0.76	0.75	0.92	1.99
SD	0.07	0.03	0.05	0.05	0.05	0.07
CV	3.36	3.19	7.25	6.89	5.51	3.67
Leiorhagium montfaouense Types (N=10)
min	1.80	1.03	0.68	0.64	0.83	1.76
max	2.30	1.16	0.81	0.77	0.99	1.99
mean	2.01	1.08	0.73	0.70	0.90	1.87
median	2.02	1.05	0.72	0.68	0.89	1.86
SD	0.15	0.05	0.05	0.04	0.06	0.09
CV	7.73	4.89	6.40	6.29	6.87	4.79

Crosse, 1872

Type species.

Crosse, 1872

sp. n.

http://zoobank.org/1C80E381-43F7-43EB-9853-425C5C6B925E

Type material.

Holotype MNHN IM 2000-27858; paratypes MNHN IM 2000-27859 (> 50), NHMW 110181 (10).

Type locality.

NeCa 12, Bouloupari: Ouaméni-valley, small stream on W-side of road in secondary forest, 21°49'46.9"S, 165°56'42.9"E, 22 May 2012.

Etymology.

The new species is dedicated to the senior author’s daughter on the occasion of her ‘quinceañera’, the 15th birthday.

Diagnosis.

sp. n. is very similar to and . It differs from both in a clearer separation of the opercular pegs and a much more delicate penis. The protoconch of the new species has more whorls than and the palatal denticle is further behind the outer lip.

Shell.

Conical, 2.2 times higher than wide, 4.5-5.5 whorls, without colour, transparent; protoconch faintly pitted with 1-1.25 whorls; palatal denticle large, elongate, c. 1/3 whorl behind outer lip; with columellar fold in the body whorl; aperture slightly higher than wide (Figs 2A, 3A,B, 4A,B).

Figure 3.

Shells (all paratypes). A, B sp. n. C, D sp. n. E, F sp. n. G sp. n. H, I sp. n.

Figure 4.

Protoconchs (left) and close-up views of apical microstructure (right). A, B sp. n. C, D sp. n. E, F sp. n. G, H I, J sp. n. Scale bars 50 µm (A, C, E, G, I), 10 µm (B, D, F, H, J).

Operculum.

Elongate-ellipsoidal, paucisprial, nucleus submarginal, orange, one large and one small non-calcareous white peg, well separated from each other (N=5) (Fig. 5A,B).

Figure 5.

Operculum. A, B sp. n. C, D sp. n. E, F sp. n. G, H sp. n.

External features.

Epidermis without pigment, eyes black.

Mantle cavity.

Ctenidium with 24–26 (2 males) or 25–28 (3 females) filaments; osphradium kidney-shaped, behind middle of ctenidium.

Digestive system.

Radula formula (N=3) (Fig. 6A): R (rhachis or central tooth): 3 1 3/2 2, L (lateral tooth): 3 1 5, M1 (inner marginal tooth): 21–25, M2 (outer marginal tooth): 27–32; stomach without caecum; rectum close to pallial oviduct in females and to prostate in males.

Figure 6.

Radula. A sp. n. B sp. n. C sp. n. D sp. n. Arrows indicate membranous junction of flank and face of lateral teeth typical for most Pacific tateid genera (partly dissolved in A and D).

Female genitalia.

Ovary without lobes, proximal end c. 1.25 whorls below apex, comprising 0.25–0.5 whorls, eventually reaching stomach; anterior capsule gland yellow-orange, posterior capsule gland opaque-white, albumen gland milky-white; proximal loop of renal oviduct upright comprising 180°, distal loop short; bursa copulatrix pear-shaped, reaching only slightly behind albumen gland; bursal duct long, entering anterior; seminal receptacle on ventral edge of and as long as bursa (N=3) (Fig. 7A).

Figure 7.

Female genitalia. A sp. n. B sp. n. C sp. n. D sp. n. ac anterior capsule gland, ag albumen gland, bc bursa copulatrix, bd bursal duct, go genital opening, od oviduct, pc posterior capsule gland, rs receptaculum seminis, vc vestibular capsule gland, ve ventral channel.

Male genitalia.

Proximal end of lobate testis 1–1.25 whorls below apex, comprising 0.75 whorls, covering proximal end of stomach; vesicula seminalis arising from anterior third of testis; penis fairly delicate with blunt end (N=2) (Fig. 8A,B).

Figure 8.

Penis. A, B sp. n. C sp. n. D sp. n. E sp. n. Scale bars = 100 µm.

Remarks.

This is sp. n. 1 of Zielske and Haase (2015). Both sp. n. and do have the columellar fold in the body whorl assumed to be unique in by Haase and Bouchet (1998). sp. n. is only known from the type locality.

Haase & Bouchet, 1998

Haase & Bouchet, 1998

http://zoobank.org/CCC4F863-76C3-44C2-A4AA-CE9DE0B726AB

Holotype IM 2000-27860; paratypes IM 2000-27861 (29), NHMW 110182 (5).

NeCa 49, Poya: Massif d’Adio, stream flowing into Grotte d’Adio, open secondary forest, 21°15'24.4"S, 165°14'46.4"E, 29 May 2012.

Other material.

NeCa 43, Poya: small stream on W-side of road between Nétéa and Goipin, on forest edge, 21°16'06.0"S, 165°14'32.0"E, 28 May 2012, -IM-2012-36075 (23), NHMW 110183 (10).

Adioincola is composed of the name of the area of Adio and the Latin noun incola meaning inhabitant, and thus refers to the type locality of the new species.

sp. n. is very similar to and sp. n. The former pair differs in penial shape, slender vs. basally broad with long terminal filament. sp. n. tends to have fewer radular denticles than . Genetically, these species differed on average at 9.65% of the positions of COI. Due PageBreakto the lack of anatomical data, both new species can only be distinguished genetically. Their COI sequences differed on average by 9.5% (p-distance).

Pupiform, 1.8 times higher than wide, 4.125-4.75 whorls, without colour, transparent; protoconch faintly pitted with c. 1 whorl; palatal denticle a small droplet 1/8 whorl behind outer lip; aperture as high as wide (Figs 2B, 3C, D, 4C, D).

Elongate-ellipsoidal, paucisprial, nucleus submarginal, orange, usually two non-calcareous white pegs, eventually accompanied by a small third one (N=3) (Fig. 5C,D).

Epidermis without pigment, eyes black.

Ctenidium with 18-19 (3 males) or 21–24 (2 females) filaments; osphradium kidney-shaped, behind middle of ctenidium.

Radula formula (N=3) (Fig. 6B): R: 4 1 4/2 2, L: 4-5 1 6, M1: 22-27, M2: 21-29; stomach without caecum; rectum close to pallial oviduct in females and to prostate in males.

Ovary without lobes, proximal end 1.25 whorls below apex, comprising 0.25-0.5 whorls, eventually reaching stomach; anterior capsule gland yellow-orange, posterior capsule gland opaque-white, albumen gland milky-white; proximal loop of renal oviduct bent forward, distal loop short; bursa copulatrix almost cubical, reaching behind albumen gland; bursal duct long, entering anterior; no seminal receptacle (N=2) (Fig. 7B).

Proximal end of lobate testis 1.25–1.5 whorls below apex, comprising 0.5-0.75 whorls, covering proximal end of stomach; vesicula seminalis arising from anterior half of testis; penis slender, terminal end occasionally forming short filament (N=3) (Fig. 8C).

This is sp. n. 4 of Zielske and Haase (2015). sp. n. occurs in the area between the villages of Nétéa and Goipin including the Massif d’Adio.

http://zoobank.org/3B015791-A03B-48BB-8C1D-1A829588B5E2

Holotype IM 2000-27862; paratypes IM 2000-27863 (28), NHMW 110184 (10).

NeCa 33, Moindou: spring-fed stream close to road in Aremu valley, under shrub, 21°35'04.8"S, 165°39'07.5"E, 26 May 2012.

The new species is named after the Aremu valley, where it has been discovered.

sp. n. is most similar to , which is, however, larger and slightly more slender, lacks the palatal denticle, and has a more massive penis. The prolonged capsule gland is unique among New Caledonian tateids. The COI sequences had a p-distance of 9.4%.

Broadly pupiform, 1.62 times higher than wide, 3.75-4.25 whorls, without colour, transparent; protoconch faintly pitted with 0.75-0.9 whorls; palatal denticle a PageBreakPageBreaksmall droplet 1/8 whorl behind outer lip; aperture practically as high as wide (Figs 2C, 3E,F, 4E,F).

Elongate-ellipsoidal, paucisprial, nucleus submarginal, orange, two non-calcareous white pegs, eventually accompanied by a small third one (N=4) (Fig. 5E, F).

Epidermis without pigment, eyes black.

Ctenidium with 15-16 (2 males) or 19-20 (2 females) filaments; osphradium elongate, slightly behind middle of ctenidium.

Radula formula (N=3) (Fig. 6C): R: 4-5 1 4-5/2-3 2-3, L: 4-5 1 4-6, M1: 26-31, M2: 20-32; stomach without caecum; rectum close to pallial oviduct in females, with short loop left of prostate in males.

Ovary without lobes, proximal end 1.25-1.75 whorls below apex, comprising 0.25-0.5 whorls, reaching stomach; capsule gland with long and slender, opaque-white vestibulum, anterior capsule gland yellow-orange, toward posterior capsule gland covered with brown spots, posterior capsule gland opaque-white with a central milky section, albumen gland milky-white; proximal loop of renal oviduct bent forward, distal loop long; bursa copulatrix higher than long, reaching behind albumen gland; bursal duct long, entering anterior; no seminal receptacle (N=3) (Fig. 7C).

Proximal end of lobate testis 1 whorl below apex, comprising c. 0.75 whorls, covering proximal end of stomach; vesicula seminalis arising from distal third of testis; penis very long and slender (N=2) (Fig. 8D).

This is sp. n. 3 of Zielske and Haase (2015). sp. n. is only known from the type locality.

http://zoobank.org/723A9EA1-CBFC-486A-AA37-69728E99AC3A

Holotype IM 2000-27865; paratypes IM 2000-27866 (3).

NeCa 30, Moindou: spring along road SW of Katrikoin, under shrub, 21°34'21.6"S, 165°41'02.5"E, 26 May 2012.

The Latin adjective clandestinus means clandestine and refers to the new species’ external identity with .

sp. n. is most similar to sp. n. and . For the distinction from sp. n. see above. Due to the lack of anatomical data, sp. n. and can only be distinguished based on 7.6% average sequence divergence of COI (p-distance).

Pupiform, 1.86 times higher than wide, 4.25-5 whorls, without colour, transparent; protoconch very faintly pitted with c. 1 whorl; palatal denticle a small droplet 1/8 whorl behind outer lip; aperture as high as wide (Figs 2D, 3G, 4G, H).

Epidermis without pigment, eyes black.

This is sp. n. 2 of Zielske and Haase (2015). sp. n. is only known from the type locality.

http://zoobank.org/7B81AF32-3FDA-49C7-A316-D84B1A5ED324

Holotype IM 2000-27867; paratypes IM 2000-27868 (20).

NeCa 44, Poya: stream at side of small road branching off road between Nétéa and Goipin toward the Vallée d’Adio, under shrub close to overgrown garden, 21°14'47.9"S, 165°15'45.0"E, 28 May 2012.

The new species is named after the village of Nétéa, which is closely proximal to our collecting locality.

sp. n. is very similar to and . In sp. n. the palatal denticle is slightly larger and 1/8 whorl further behind the outher lip. The operculum has only a single denticle compared to 2-3 in and . The distal loop of the renal oviduct of the new species forms a 270° loop counter-clockwise, whereas in the other two species this part of the oviduct is bent PageBreak180° clockwise. The penis of sp. n. is long and slender in contrast to the other species, where it has a broad base and a very long filament.

Elongate-pupiform, 1.88 times higher than wide, 4.25–5 whorls, without colour, transparent; protoconch faintly pitted with c. 1 whorl; palatal denticle an elongate droplet c. 1/4 whorl behind outer lip; aperture slightly wider than high (Figs 2E, 3H, I, 4I, J).

Elongate-ellipsoidal, paucisprial, nucleus submarginal, orange, one non-calcareous white peg (N=4) (Fig. 5G, H).

Epidermis without pigment, eyes black.

Ctenidium with 15 (1 male) or 19-22 (5 females) filaments; osphradium short-elongate, behind middle of ctenidium.

Radula formula (N=4) (Fig. 6D): R: 4 1 4/2-3 2-3, L: 4-5 1 5, M1: 20-25, M2: 24-27; stomach without caecum; rectum close to pallial oviduct in females, with short loop left of prostate in male.

Ovary without lobes, proximal end 1.25-1.5 whorls below apex, comprising 0.25-0.5 whorls, not reaching stomach; anterior capsule gland yellow-orange, posterior capsule gland opaque-white, albumen gland milky-white; proximal loop of renal oviduct bent forward, distal loop short; bursa copulatrix globular, reaching slightly behind albumen gland; bursal duct long, entering anterior; no seminal receptacle (Fig. 7D).

Proximal end of lobate testis 1 whorl below apex, comprising slightly more than 0.5 whorls, covering proximal end of stomach; vesicula seminalis arising approximately in middle of testis; penis very long and slender (N=1) (Fig. 8E).

This is sp. n. 5 of Zielske and Haase (2015). sp. n. is only known from the type locality.

Morphometry

The CVA plot (Fig. 9) comparing species of shows the high variability of . The associated MANOVA was highly significant (Wilk’s λ = 0.062, DF1 = 35, DF2 = 490.4, F = 13.16, p = < 0.001). Many pairwise comparisons of populations were significant as well (Table 3). and sp. n. fell within the variation of . According to the CVA, they were not more different from each other than from populations of . Assignment and jacknifed assignment tests allocated 80 (62.5%) and 67 (52.3%) of a total of 128 shells to their original sample indicating the considerable overlap of shapes.

Figure 9.

CVA plot for . Samples without numbers are paratypes.

Table 3.

Pairwise morphometric comparisons of samples. Hotelling’s T2 tests, based on five shell measures; significance assessed after sequential Bonferroni correction; sample sizes are given in Table 2. *; NS.

p < 0.05

, not significant

	1	2	3	4	5	6	7
1 Hemistomia andreae
2 Hemistomia cockerelli Types	*
3 Hemistomia cockerelli NeCa11	*	*
4 Hemistomia cockerelli NeCa17	*	*	*
5 Hemistomia cockerelli NeCa21	NS	*	*	NS
6 Hemistomia cockerelli NeCa36	*	*	*	NS	NS
7 Hemistomia cockerelli NeCa54	*	*	*	NS	*	NS
8 Hemistomia nyo NeCa35	*	*	*	NS	NS	*	NS

The CVA (Fig. 10) for revealed species clusters with sp. n. and sp. n. overlapping with and sp. n. largely grouping with and . The MANOVA was again highly significant (Wilk’s λ = 0.009, DF1 = 50, DF2 = 669.2, F = 23.56, p = < 0.001), as were most pairwise comparisons (Table 4). Note that comparisons involving sp. n. or were less meaningful because of the small sample sizes. Assignment and jacknifed assignment tests performed similar as for with only 103 (64.0%) and 88 (54.7%) correctly allocated shells of a total of 161.

Figure 10.

CVA plot for . Samples without numbers are paratypes.

Table 4.

Pairwise morphometric comparisons of samples. Hotelling’s T2 tests, based on five shell measures; significance assessed after sequential Bonferroni correction; sample sizes are given in Table 2. *; NS.

p < 0.05

, not significant

	1	2	3	4	5	6	7	8	9	10
1 Leiorhagium adioincola NeCa49
2 Leiorhagium aremuum	*
3 Leiorhagium clandestinum	NS	*
4 Leiorhagium neteae	*	*	*
5 Leiorhagium ajie Types	*	*	NS	*
6 Leiorhagium douii Types	*	*	*	*	*
7 Leiorhagium kavuneva Types	*	*	NS	*	*	*
8 Leiorhagium kavuneva NeCa15B	NS	*	NS	*	*	*	*
9 Leiorhagium kavuneva NeCa29	NS	*	NS	*	*	*	*	NS
10 Leiorhagium monachum Types	*	*	NS	*	NS	NS	*	*	*
11 Leiorhagium montfaouense Types	*	*	*	*	*	NS	*	*	*	NS

Phylogenetic analysis

In the phylogenetic analysis (Fig. 11), and were sister groups, both with 100% bootstrap support. Within , the elongate-pupiform species , and sp. n. were paraphyletic with respect to the more conical-pupiform species, which received a bootstrap support of 91%. Otherwise, relationships among species of were not well supported. All four new species were (phylo)genetically well distinct as indicated by the branch lengths expressing genetic distances. Within , the picture was very similar with well differentiated species but otherwise little resolution. Average pairwise uncorrected genetic distances based on the COI-fragment were ≥ 7.4% and are summarized in Table 5.

Figure 11.

Maximum likelihood phylogram showing bootstrap support when > 50%. Outgroup pruned from tree; new species highlighted by bold face type.

Table 5.

Average pairwise uncorrected (p) distances between selected species based on the COI-fragment (in %).

	1	2
1 Hemistomia andreae
2 Hemistomia cockerelli	8.6
3 Hemistomia nyo	8.8	9.5
	1	2	3	4
1 Leiorhagium adioincola
2 Leiorhagium ajie	9.3
3 Leiorhagium aremuum	10.6	9.4
4 Leiorhagium clandestinum	9.5	7.8	7.4
5 Leiorhagium kavuneva	9.7	8.1	8.5	7.6

Discussion

Our phylogenetic analyses based on DNA sequence data confirmed the suspicion of Haase and Bouchet (1998) that additional cryptic species in this snail fauna will be identified once molecular methods are applied emphasizing the huge morphological variability of certain nominal species. Recent accounts on tateid gastropods from Vanuatu and Fiji (Zielske and Haase 2014a, b) have revealed extensive radiations of morphologically very similar species. However, in contrast to the New Caledonian taxa, the radiations on those archipelagos are comparatively young (Zielske and Haase 2015). Four of the five species described here are hardly distinguishable from known taxa based on measurements despite being genetically well differentiated with even uncorrected distances (see Fregin et al. 2012) of at least 7.4% to their next similar congeners. Whether this means that morphologically similar species occupy similar niches is impossible to tell at this stage because the relationship of shell morphology to habitat has not been investigated among truncatelloidean gastropods except for a few accounts on (Haase 2003, Holomuzki and Biggs 2006, Kistner and Dybdahl 2013). Although ranges overlap or are contiguous, sibling species have not (yet) been encountered in sympatry, i.e. in the same spring or stream.

The new species provide an additional truncatelloid example stressing the importance of an integrative taxonomic approach combining morphological, anatomical and genetic methods (e.g. Haase et al. 2007, Delicado and Ramos 2012). Given the mosaic nature of evolution of these small gastropods with morphologically as well as genetically cryptic species (e.g., Haase et al. 2007, Haase 2008, Zielske et al. 2011, Delicado and Ramos 2012, Liu et al. 2013), we do not adhere to a fairly strict scheme of species identification as advocated elaborately e.g. by Schlick-Steiner et al. (2010). Instead we advocate the approach of Padial and de la Riva (2010) who have a more natural vision of the evolutionary processes potentially involved in speciation. For instance, they acknowledge that the congruence of different character sets, pivotal for taxonomic decisions for Schlick-Steiner et al. (2010), may be plesiomorphic.

Genetic differentiation was an important indicator of species status. Pairwise p-distances > 7.4% are far above any threshold suggested by advocates of barcoding (e.g., Hebert et al. 2003, 2004; Ratnasingham and Hebert 2007). However, again we do not adhere to a strict scheme as there may be no mitochondrial differentiation between good species as well as considerable variation within species of spring snails (e.g. Haase 2008; Zielske et al. 2014a; see also Fregin et al. 2012). That genetic differentiation does reflect species status for the new taxa is also indicated by the comparison of their branch lengths to branch lengths among morphologically well defined species in our phylogenetic analysis.

While conducting our morphometric analyses we appreciated that the measuring methods applied for the material described previously (Haase and Bouchet 1998) and for this account are incompatible. Obviously, using an ocular micrometer fitted to a dissecting microscope produced inaccurate data, although the measurements were quite consistent judging from the fairly low coefficients of variation, which were of a PageBreaksimilar order of magnitude as those computed for the present data. Therefore, we had to re-measure the old samples used in our comparisons.

Another methodological problem almost expectedly occurred in the field. All collections made for our previous monograph (Haase and Bouchet 1998) were geo-referenced from maps. This proved to be fairly inaccurate when we tried to relocate sites in 2012 guided by GPS. Additional difficulties arose from recent road development and land-use changes. Many villages are now accessible on much broader roads than 20 years ago. Construction has obviously destroyed small road-side springs and seepages and changed the course of streams. Other sites were destroyed by extensive fires affecting entire valleys or hills. , in our analysis part of the outgroup, used to be common when first collected in 1992. Now we found only a few specimens. It remains to be seen whether there are other (extant) populations in the unexplored hinterland of Boyen. In contrast, , collected in a few seepages on Col d’Amoss in the far Northeast in 1989, is now probably extinct. The entire area has lost its primary vegetation. Today, the fire resistant niaouli () and shrubland are dominating and streams harbor a very depauperate fauna.

Four of the five new species were found in single sites and the fifth was found at only two sites. Considering the vulnerability of small habitats like springs and the rapid anthropogenic development and changes on New Caledonia just outlined immediately raises concern regarding the chances of long-term survival of these species (see also Haase et al. 2010). Most sites we surveyed were rather easily accessible, close to roads, so that one can assume that there are other populations deeper in the forests or forest remnants. Nevertheless, given that the area of occupancy of each species is certainly less than 10 km2, that ranges of spring snails are almost naturally severely fragmented, and the rapidly progressing change of land cover, areas of occupancy as well as habitat, hence the numbers of populations will decline. Therefore, all five species and probably the majority of New Caledonian tateids qualify as critically endangered according to the criteria (CE, B2,a,II-IV) of the International Union for Conservation of Nature (IUCN 2012).