Generic switch-over during ontogenesis in Dimorphacanthella gen. n. (Collembola, Isotomidae) with barcoding evidence

Abstract A new genus Dimorphacanthella is established for Tetracanthella anommatos Chen and Yin, 1984 and Dimorphacanthella mediaseta sp. n. from China. The new genus exhibits an unusual metamorphosis: small juveniles, previously called Uzelia anommatos Yue & Yin, 1999 get the second pair of anal spines resulted from moulting and become “Tetracanthella”. Species identity of forms with two and four anal spines is proved by barcoding analysis. The derivation of anal spines is compared among genera having four anal spines.


Introduction
With this paper we continue our study of Collembola of family Isotomidae from China Gao and Potapov in press;Huang et al. 2009Huang et al. , 2010. After recent revisions of the genus Tetracanthella Schött, 1891 one species Tetracanthella anommatos Chen and Yin, 1984 remained curious (Deharveng 1987;Potapov 2001). Except possessing four anal spines, this odd species has no pigment or ocelli and exhibits an uncommon number and arrangement of sensilla on the body (Chen and Yin 1984). Its generic position has not been fully decided. During our study of the family Isotomidae of China we had an opportunity to collect this species from type (Shanghai) and other localities. In several populations T. anommatos was mixed with another unusual blind form which was identifi ed by us as Uzelia anommatos Yue & Yin, 1999, which was also described from Shanghai area. Th e latter one shares several important characters with T. anommatos and was always recorded by us in the juvenile form. Th is led us to re-describe these two species using the modern morphological knowledge and to test their independence by barcoding analysis. In our collection from Northwest China (Ningxia Province), another species closely related to T. anommatos was found. In this paper we establish a new genus, having uncommon metamorphosis during ontogenesis, re-describe T. anommatos, and describe a new species.
Type species: Dimorphacanthella anommatos (Chen & Yin), 1984, comb. n. Remarks: Sharing four anal spines on genital segment, the new genus Dimorphacanthella formally resembles three genera namely Tetracanthella, Blissia Rusek, 1985 andSibiracanthella Potapov &Stebaeva, 1995. All four genera also have the same derivation of inner anal spines which are, following the notation of Deharveng (1978), p1-setae modifi ed. Outer anal spines derived from p2-setae in Tetracanthella (Fig. 14) and Blissia and a1-setae in Sibiracanthella. Outer anal spines of Dimorphacanthella are modifi ed setae positioned in posterior part of Abd. V and between two macrosetae pp1 and pp2. Since the designation of setae of Abd. V is not developed enough we notate these setae as setae x on the schemes (Fig. 15). Seta x is a ordinary seta in late instars of Tetracanthella, while in Dimorphacanthella it is modifi ed to a spine at later instars and absent at early instars . Seta p2 which is a lateral spine in Tetracanthella remains unmodifi ed in Dimorphacanthella and is located anterior to and between sensilla s1 and s2. Th e new genus has a pair of ordinary setae in front of median spines in late instars (notated as m1 seta by us) (Figs 15). Th ese setae were never seen in other genera having spines in p1-position. Th ese and other diff erentiated characters of genera with four anal spines are presented in Table 1.
Blindness and presence of unpaired seta on Abd. IV are only shared with Martynovella nana nana (Martynova, 1967) described from Central Asia. Th is species, however, has two anal spines at all age instars, more complete sensillary set, lacking seta m1 in front of spines, and other less signifi cant diff erentiating features. Another subspecies, Martynovella nana kirgisica (Martynova, 1967) has no unpaired seta on Abd. IV. Th e number and homology of real anal spines are of high value in generic taxonomy of Anurophorinae and appeared to be constant in the course of postembryonic ontogenesis (Deharveng 1978), except epitokous and ecomorphic forms of Proisotoma Börner, 1901and Cliforga Wray, 1952(Fjellberg 1988Najt 1983). In the family, Dimorphacanthella anommatos is the only case where the real anal spines on high papillae appear after moulting.
Etymology: Th e name of the new genus refers two morphs, juvenile and adult, having diff erent number of anal spines in at least one species of the genus.
Age dependent morphology: Small individuals with up to 0.65 mm length have fewer axial setae, 2 postlabial setae, and two anal spines so then corresponding to morphology of Uzelia anommatos. General appearance of fully grown and young individuals is shown in Figs 3-4.
Remarks. For the diff erence from D. mediaseta see the remarks section.  Description. Size of adult males and subadult females up to 1.0 mm (adult female not seen). Body shape slender, general appearance somewhat that of the genus Stenaphorura (Absolon, 1900) (Onychuridae). No pigmentation. Body with well visible reticulation, no elongated polygons. With 4 strongly chitinized anal spines arranged almost in one transversal row at the end of abdomen. Inner anal spines 1.1-1.4 times longer then outer spines. Ocelli absent. PAO narrowly elliptical, with weak constriction, 1.8-2.2 as long as unguis 3. Outer mouth parts as in previous species, labial palp shown in Fig. 9. Maxillary claw strong, lamellae not beyond its tip. Ventral side of a head with 3+3 postlabial setae. Ant. I with 11 (rarely with 10) setae, 2 bms, dorsal and ventral, and 2 ventral s, one of them curved and 3-4 times longer than another. Ant. II normally with 17 setae, 3 bms and one curved s. Ant. III without bms and with 5 s, inner pair of sensilla of AO much shorter and set in cuticular groove (Fig. 8). Several differentiated sensilla on Ant. IV, subapical organite small, subapical microsensillum short.
Remarks. Th e new species diff ers from D. anommatos by presence of medial pair of macrosetae on the fi rst three abdominal tergites. Sensilla on tergites, especially lateral pair on Abd. V, are thinner than in D. anommatos. Seta p2 on Abd. V also sharply discriminates these two species (in more posterior position in D. mediaseta, Figs 1 and 10). Number of anal spines in small juvenile individuals is unknown. Name derivation. Th e name refers to medial macrosetae on body diff erentiating the new species.
Barcoding analysis. DNA barcoding sequence was proved very effi cient for characterising Collembolan species (Hogg and Hebert 2004). Here, we used barcode in order to validate those two forms of D. anommatos are really belong to the same species, in spite of strong morphological diff erences. Collembolan specimens. Collembolan species for DNA barcoding analysis were collected from Zhongjia Mountain, 31°05'N, 121° 09'E, 25 m alt., Songjiang county, Shanghai city in 75% ethanol by the Tullgren funnel method. Th ey were stored in 100% ethanol at -20°C after morphological identifi cation. We barcoded separately 2 individuals of 'T. anommatos' with four anal spines and 4 individuals of 'U. anommatos' with two anal spines.
DNA extraction, amplifi cation and sequencing. Genomic DNA was extracted from one individual using the Wizard SV Genomic DNA Purifi cation System (# 2361). Th e mitochondria COI gene sequence was amplifi ed (658 bp) by primer pair LCO (5' -GGTCAACAAATCATAAAGATATTGG-3') / HCO (5'-TAAACTTCAGGGT-GACCAAAAAATCA-3') (modifi ed from Folmer et al. 1994). We use the following profi le: 94°C initial denaturing for 4 min; 10 cycles of 94°C denaturing for 30 s, 45°C annealing for 30 s, and 72°C extension for 1 min and 30 s; then, with 25 cycles of 94°C denaturing for 30 s, 50°C annealing for 30 s, and 72°C extension for 1 min and 30 s; and a fi nal extension at 72°C for 8 min. PCR products were purifi ed and then were sequenced directly using both of the amplifi cation primers.
Results. Six sequences (length 658 bp) were obtained from 6 individuals. Th e two individuals of the four-spined form showed the same sequence, as the four individuals of the two-spined form. Th is suggested that the two forms are a same species in agreement with our morphological result. GenBank Accession number of barcoding sequences are HM366600 and HM366601.