One new species of the family Niphargidae from Bosnia and Herzegovina ( Contribution to the Knowledge of the Amphipoda 289 )

From the subterranean waters of the Velika Bukovička špilja Cave near Bukovica village (Tomislavgrad region, Bosnia and Herzegovina) is described and figured one new species of the family Niphargidae (Amphipoda, Gammaridea), Niphargus bukovicensis, sp. n. This species is characterized by presence of elevated number of spines on dactylus of pereopods 6 and 7 only, by numerous setae along dactylus outer margin of gnathopods 1-2, pointed epimeral plate 3 and by elongated inner and outer ramus of uropod 3 in males. The taxonomical relation of this species regarding other similar members of genus Niphargus Schiödte, 1849, is discussed.


Introduction
The fauna of the Amphipoda in Bosnia and Herzegovina has been studied by various authors and over 60 species of various families (Gammaridae, Niphargidae, Bogidiellidae, Hadziidae, Typhlogammaridae, Crangonyctidae, Talitridae, etc.) have been established, including nearly 30 members of the genus Niphargus Schiödte, 1849 (G.Karaman, 2010); over half of known Niphargus taxa in Bosnia and Herzegovina are endemic for this and adjacent regions.
During the recent investigations of caves in Bosnia and Herzegovina, organized by the speleological Society Mijatovi Dvori in Tomislavgrad, in association with the speleologists from Bosnia and Herzegovina, Croatia, Montenegro and other countries, various samples of Amphipoda have been collected and gave me at disposition for study.During the study of this material, one new species of the family NIphargidae (Amphipoda Gammaridea) is established and described here.
The existence of over 300 known taxa of genus Niphargus from Europe, Asia Minor and Near East request more and more detailed descriptions of each species to make possible recognition of each species and its variability, and eventual comparison of morphological characters with results of genetic investigations of these species.

Material and Methods
The sample was collected in the subterranean water by hand-net and catching by hands.The collected specimens were preserved in 70% ethanol.Specimens were examined and dissected using a Wild M 20 stereomicroscope and drawn using a camera lucida attachment.The animals were temporarily mounted in the mixture of glycerin and water for dissection and drawing of body parts, and later transferred on slides with liquid of Faure for final preservation.
The body length of examined specimens was measured by tracing individual's mid-trunk lengths (tip of the rostrum to end of telson) and drawings were made using a camera lucida and inked manually.Some morphological terminology and setae formulae follow G. Karaman's terminology (Karaman, G., 1969;1970;2012b) for palpus article 3 of mandible [A-setae= setae on outer face; B-setae= setae on inner face of article; D-setae= short setae along ventral lateral margin of article; F-setae= long setae at distal part of article], and distal corner of propodus of gnathopods 1 and 2 [S-spine= corner palmar spine on outer face; L-spines= slender serrate spines sitting near S-spine on outer face; M-setae= row of long facial submarginal setae on outer face; R-spine= subcorner spine on inner face].This study is based on the morphological, ecological and zoogeographical data.

Diagnosis
Body very slender, with short antennae and legs, and with long uropod 3. Epimeral plates 1 and 2 with almost subrounded ventroposterior corner, epimeral plate 3 with pointed corner and concave posterior margin.
Maxilla 1 inner plate with 2-3 setae, outer plate with 7 spines (most of them with one lateral tooth each); Inner plate of maxilliped short, with 3 distal spines.Antenna 2 flagellum longer than last peduncular article.
Head with short rostrum and short subrounded lateral cephalic lobes, ventroanterior excavation well developed eyes absent (fig.1E).
Antenna 2: peduncular article 3 short, with distoventral bunch of setae; peduncular articles 4 and 5 of equal length; article 4 at ventral margin with 3-4 bunches of setae (the longest setae exceeding the diameter of article itself (fig.1D), at dorsal margin with 4 bunches of short setae; article 5 at ventral margin with 5 bunches of long setae (setae remarkably exceeding the diameter of article itself), at dorsal margin with several bunches of short setae; flagellum slender, with missing distal part, probably longer than last peduncular article (fig.1D) (see sub Variability, fig.6C).Antennal gland cone short (fig.1D, 6C).
Maxilla 2: both plates with distolateral setae only (fig.3G).Maxilliped: inner plate short, with 3 distal pointed spines mixed with single setae (fig.3F); outer plate reaching nearly half of palpus article 2 and provided with row of distomarginal lateromarginal spines; palpus article 3 at outer margin with one median and one distal bunch of setae; palpus article 4 at outer margin with 2 median setae, at inner margin with 2 setae near basis of the nail (fig.3F), nail is much shorter than pedestal.
Gnathopod 2: article 2 along anterior and posterior margin with numerous long setae; article 3 at posterior margin with one bunch of setae; article 5 shorter than propodus (ratio: 40:50), along anterior margin with one median and one distal bunch of setae (fig.2D).Propodus trapezoid, only slightly longer than broad (ratio: 98:90), along posterior margin with 9 transverse rows of setae (fig.2E); palm slightly convex, inclined hardly over half of propodus-length, defined on outer face by one corner S-spine accompanied laterally by 2 L-spines and 4 facial long M-setae, on inner face by one subcorner R-spine (fig.2F).Dactylus reaching posterior margin of propodus, along outer margin with 10 single or paired median setae, along inner margin with 6-8 short setae (fig.2E).
Pereopods 3 and 4 relatively short.Pereopod 3: article 2 at anteroproximal margin with long setae, at anterodistal margin with short setae (fig.3A), posterior margin with row of long setae.Articles 4-6 of unequal length (ratio: 56:40:45); article 4 at posterior margin with 5 bunches of setae (the longest setae exceeding the diameter if the article itself), along anterior margin with 3 bunches of setae; article 5 at posterior margin with 3 bunches of longer setae, along anterior margin with 2 bunches of short setae; article 6 at posterior margin with 5 bunches of short spines or single short setae, along anterior margin with 3 bunches of setae.Dactylus strong, much shorter than article 6 (ratio: 15:45), at inner margin with one strong spine and one short seta near basis of the nail (fig.3B), along outer margin with one short median plumose seta; nail shorter than pedestal (ratio: 29:36).
Pereopod 4: article 2 at anterodistal margin with row of short setae and at anteroproximal margin with several long setae (fig.3C); posterior margin of article 2 with row of long setae; articles 4-6 of unequal length (ratio: 51:38:44); article 4 along posterior margin with 4 bunches of setae (the longest setae are nearly as long as diameter of the article), along anterior margin with 3 groups of setae; article 5 along posterior margin with 2 bunches of short setae mixed with 2 strong spines.Article 6 along posterior margin with 5 groups of short spines or setae, along anterior margin with 3 bunches of setae.Dactylus strong, much shorter than article 6 (ratio: 16:44), along inner margin with one strong spine and short seta near basis of the nail, along outer margin with one median plumose seta (fig.3D); nail shorter than pedestal (ratio: 27:36) (fig.3D).
Pereopod 5: article 2 rather longer than broad (ratio: 77:50), along anterior rather convex margin appears a row of 7 single or group of spine-like setae (fig.4A), along posterior poorly concave margin are attached nearly 17 short setae, ventroposterior dilatation not forming the lobe (fig.4A).Articles 4-6 of unequal length (ratio: 51:53:53); article 4 at anterior margin with 4 bunches of short setae, along posterior margin with 3 groups of single spines or setae; articles 5 and 6 along both margins with several bunches of short spines mixed with single short setae.Article 2 is longer than article 6 (ratio: 77:53).Dactylus is short and strong, much shorter than article 6 (ratio: 17:53), along inner margin with one strong spine and short seta near basis of the nail, along outer margin with one median plumose seta (fig.4B); nail shorter than pedestal (ratio: 27:42).
Uropod 1 slender, peduncle without distal tubercle, bearing dorsoexternal row of spines and dorsointernal row of setae (except distal spine); inner ramus elongated, as long as peduncle, slightly curved distally, bearing one lateral and 3-4 distal short spines, as well as 5 bunches of simple short setae (fig.5E); outer ramus shorter than half of inner ramus, bearing 4 distal short spines and 3 lateral bunches of short setae.
Uropod 3 long, with elongated peduncle and inner ramus.Left uropod 3: peduncle much longer than broad (ratio: 54:18) bearing one lateral spine and lateral and distal short setae or spines (fig.5F).Inner ramus slightly shorter than peduncle (ratio: 40:54), bearing several lateral spines and lateral and distal short simple setae (fig.5F).Outer ramus 2-articulated, slender: first article poorly longer than second article (ratio: 133:120), along outer margin with 6 bunches of short simple setae, along inner margin with bunches of single spines and short simple setae accompanied with longer plumose setae (fig.5F); second article along both margins and tip provided with bunches of simple setae.
Right uropod 3: peduncle like that of left uropod 3. Inner ramus nearly as long as peduncle, bearing 3 lateral short spines and several lateral and distal short simple setae (fig.5G).Outer ramus 2-articulated, first article slightly longer than second article (ratio:136:113), along inner ramus with 7 groups of short spines and 1-2 simple short setae, accompanied by single longer plumose setae (fig.5G), along outer margin appear 5 groups of short simple setae; second article along both margins and tip with several groups of short simple setae.
Telson slightly longer than broad (ratio: 79:70), incised nearly 2/3 of telson-length; lobes slightly tapering distally, provided with 3 short distal spines and 2 facial spines; along outer margin is attached one spine and 0-1 seta, along inner margin of each lobe appear 2-3 short spines; a pair of short plumose setae is attached near the middle of outer margin of each lobe (fig.6B).
Antenna 1 reaching half of body-length, main flagellum consisting of 27-28 articles.Antenna 2: articles 4 and 5 like these in male, flagellum longer than last peduncular article and consisting of 13 articles.
Maxilliped inner plate with 3 distal spines; palpus article 3 at outer margin with one median and one distal group of setae; palpus article 4 at inner margin with 2 setae near basis of the nail, along outer margin with 1-2 median setae.
Gnathopod 2: article 2 along anterior margin with row of nearly 10 shorter setae, along posterior margin with several bunches of long setae (fig.7D); article 3 at posterior margin with one bunch of setae; article 5 nearly as long as propodus, along anterior margin with 2 bunches of setae.Propodus trapezoid, nearly as long as broad as or hardly broader than long, along posterior margin with 11 transverse groups of setae (fig.7E).Palm slightly convex, inclined nearly half of propodus-length, defined on outer face by 1 S spine accompanied laterally by 2 L-spines and 4 long facial M-setae (fig.7E), on inner face by one R-spine.
Dactylus reaching posterior margin of propodus, along outer margin with row of 9 long setae, along inner margin with row of short setae.
Pereopods 3 and 4 moderately setose.Pereopod 3: anteroproximal margin of article 2 with several long setae, at anterodistal margin with row of short setae, along posterior margin with numerous long setae (fig.6D).Articles 4-6 of unequal length (ratio: 50:40:41); article 4 at posterior margin with 3 bunches of setae (the longest setae exceeding the diameter of article itself), along anterior margin with 3 bunches of shorter setae.Article 5 at posterior margin with one median spine and 3 bunches of setae (fig.6D); article 6 along posterior margin with 5 single or paired short spines and single short seta, along anterior margin with 3 bunches of short setae.Dactylus much shorter than article 6 (ratio: 17:41), along inner margin with one spine and short seta near basis of the nail, along outer margin with one median plumose seta (fig.6E); nail is shorter than pedestal (ratio: 24:30).
Pereopod 4: article 2 at anterodistal margin with row of shorter setae and at anteroproximal margin with several long setae, and with numerous long setae along posterior margin (fig.6F); articles 4-6 of unequal length (ratio: 45:34:41); article 2 at posterior margin with 3 bunches of setae as long as or poorly longer than diameter of article itself, along anterior margin with 3 groups of setae; article 5 at posterior margin with 2 spines and several short setae; article 6 along posterior margin with 4-5 groups of short setae or single spines.Dactylus much shorter than article 6 (ratio: 13:41), along inner margin with one strong spine and short seta near the basis of the nail, at outer margin with one median plumose seta (fig.6G); nail shorter than pedestal (ratio.24:26).
Pereopod 6: article 2 much longer than broad (ratio: 85:46), along anterior poorly convex margin with 6 groups of spine-like setae, along posterior almost straight margin with nearly 15 short setae, ventroposterior dilatation not forming the lobe (fig.8C).Articles 4-6 of unequal length (ratio: 60:70:83); article 4 at anterior margin with 4 groups of short setae, along posterior margin with 4 groups of spines and one long seta; article 5 and article 6 along both margins with groups of short spines accompanied sometimes with setae.Article 2 is almost as long as article 6. Dactylus much shorter than article 6 (ratio: 22:83), along inner margin with 1 spine and 1 short seta, at outer margin with 1 median plumose seta (fig.8D); nail is shorter than pedestal (ratio: 28:53).
Uropod 3: peduncle longer than broad (ratio: 46:22), bearing 2 lateral and several distal short spines and short setae (fig.9E); inner ramus much shorter than peduncle (ratio: 46:18), scale-like, with lateral spine and simple seta and with distal spine and several simple setae (fig.9E); outer ramus 2-articulated, first article along outer ramus with 4 groups of short spines and single simple setae, along inner margin with 5 groups of short spines and single short simple setae accompanied by single long plumose setae; second article much shorter than first article (ratio: 49:118), along both margins and tip with groups of simple setae (fig.9E).
Telson rather longer than broad (ratio: 82:68), incised nearly 2/3 of telson-length; each lobe with 3-4 distal and 2-3 facial spines; along inner margin of lobes appear 1-2 spines; along outer margin of each lobe are attached 0-1 spine (fig.9A); a pair of short plumose setae is attached near the middle of outer margin on each lobe.

Variability.
In our sample, the adult females with setose oostegites were 11.0-14.0mm long, and males were up to 20.0 mm long.
Inner plate of maxilla 1 bearing 2-3 setae, 6 spines of outer plate in maxilla 1 usually with one lateral tooth, rarely one spine is provided with 2 lateral spines, inner spine is always with several lateral teeth.
Along inner margin of dactylus on pereopods 3-5 appears always one spine near basis of the nail, in males and females.Dactylus of pereopod 6 is mainly provided at inner margin with one spine, sometimes with 2 spines (male 12.2 mm; female ovig.13.0 mm).
In male 15.0 mm antenna 2 exceeding the length of last peduncular article and consisting of 13 articles (fig.6C).
Evidently the males are more inclined to have more spines on dactylus of pereopod 7 than the females, but the increasing number of these spines is not in direct correlation with the size of the specimens.Propodus of gnathopods 1-2 is hardly narrower in female of 12.0 mm: gnathopod 1 propodus is longer than broad (ratio: 87: 76) (fig.9F), gnathopod 2 propodus is longer than broad also (ratio: 92:85) (fig.9G).
In adult males is inner ramus of uropod 3 elongated, reaching nearly the size of peduncle.Similar shape of uropod 1 is present in Niphargus karamani Schellenberg, 1935 known from Croatian-Slovenian border [loc.typ.: well near Miljana castle (on riverbank of Sutla river, Sava tributary), Croatia [S.Karaman, 1950;G. Karaman, 2014).Derivatio nominis.The name "bukovicensis" arrives from the name of the locality Bukovica village, where the species was collected.

Remarks and affinities
Niphargus bukovicensis is characterized by presence of additional spines on dactylus of pereopods 6 and 7.The presence of additional spines on dactylus of various pereopods is present in various taxa of genus Niphargus: in some cases these presence of additional spines can be more occasional than regular (Niphargus navotinus G. Karaman, 2014a) or always occasional (Niphargus longicaudatus Costa, 1851), or can be more or less regular (N.stankoi G. Karaman, 1974, N. radzai G. Karaman, 2014a, N. castellanus S. Karaman, 1960, etc.).
Based on the presence of additional spines on dactylus of some pereopods, N. bukovicensis is rather similar to the members of the Supraniphargus Group of species (S.Karaman, 1950), characterized by elevated number of spines on some or all pereopods.On the other hands, the existence in our sample of adult specimens with and without additional spines on dactylus of pereopods 6 and 7, show the transitive position of Supraniphargus group regarding some other groups of Niphargus taxa.
In the central Europe is present Niphargus tatrensis Wrzesniowski 1890-Complex of taxa [loc.typ.: Zakopane, Poland], characterized also by presence of some additional spines of some of pereopod dactyl.Various forms and taxa of this complex have been described; most of them probably are a distinct taxa.This Complex of taxa is still poorly known and its relation to the western Balkan taxa and to N. illidzensis Complex is poorly known and needs detailed studies.
Further studies on all members of species with additional spines on dactylus of pereopods will put more light on the taxonomical value of this taxonomical character in various members of Supraniphargus Complex and its relation to other Niphargus taxa.

Conclusions
The discovery of new species Niphargus bukovicensis, sp.n. in the Velika Bukovička špilja Cave in Bosnia and Herzegovina show the existence of high number of endemic Niphargus taxa in the subterranean waters of Bosnia and Herzegovina and its connections to the taxa of adjacent regions, thanks to the geological, ecological, zoogeographical and other events in the past on this territory, and the present condition of this region.
The members of the family Niphargidae are still in the process of speciation and splitting into numerous more or less distinct populations.Among them, some of these populations developed complete reproductive isolation (genetic or geographic) forming a distinct species.
On the other hands, many populations are still in the process of isolation and transition, still in some manner connected together.In this light, to fix the boundaries between single taxa is very difficult and undergoes often towards subjective criteria.
Neither pure morphological studies, nor pure genetical studies can resolve alone this problem; it is necessary to use of combination of various methods to resolve this problem, leaving still questionable results.We don`t know the real value of character appearance of additional spines on dactylus, as a character used to separate the single taxa, but in any case, this character must be present in the genetic code of these populations, and maybe next deep genetical and molecular studies will recognize it as a distinct character.
The number of splitting populations into a distinct taxa will depend on how deep we will go in establishing of differences among them, and of our decisions where to put the border under which the further splitting of a distinct taxa begin to be useless (G.Karaman 2012a: 33).