Edaphologia, No. 104: 11–18, March 29, 2019 11 Juvenile Desoria gracilliseta (Collembola, Isotomidae) with four anal spines Naoyuki Matsumoto1, Yasuhiko Suma2, and Taizo Nakamori3 16-22-2 Bunkyo, Chitose 066-0052, Japan 26-7-32 Harutori, Kushiro 085-0813, Japan 3Graduate School of Environmental and Information Sciences, Yokohama National University, 79-7 Tokiwadai, Hodogaya, Yokohama 240-8501, Japan Corresponding author: Naoyuki Matsumoto ([email protected]) Received 19 June 2018; Accepted 1 November 2018 Abstract Desoria gracilliseta colonizes a fruticose lichen, Cladonia sp., in a snowy habitat in Hokkaido, Japan. Ju- veniles appear in October, continue to develop under snow, and mature in May. Recent surveys from mid-September to mid-October revealed the presence of a Tetracanthura-like collembolan, in addition to D. gracilliseta juveniles. Body lengths of the Tetracanthura-like collembolan remained consistent throughout the duration of the surveys averaging 0.49 mm, whereas D. gracilliseta juveniles grew steadily from 0.54 mm to 0.8 mm. Both forms were almost indis- tinguishable except that the Tetracanthura-like collembolan had four anal spines on abdominal segment VI and short spines on the head and thorax segment II. Large individuals distinctive of D. gracilliseta collected in March were also included for comparisons in morphology and DNA sequences. Antennal segment IV and the furca developed with in- creasing body length. DNA barcode analysis, using mitochondrial cytochrome c oxidase subunit 1 and 16S ribosomal RNA genes confirmed the genetic identities of the Tetracanthura-like collembolan, and juveniles and subadults of D. gracilliseta to be D. gracilliseta. The significance of morphological changes in D. gracilliseta was discussed in terms of survival strategy against predation. Key words: Anal spine, Desoria gracilliseta, DNA barcoding, morphological change, Tetracanthura sp. The taxonomic entity of Tetracanthura is regarded as Introduction dubious. Juveniles possessing anal spines have been found The collembolan Desoria gracilliseta (Börner, 1909) is in Isotoma (Najt, 1983), and members of Tetracanthura are active even under snow and univoltine on a fruticose lichen considered as juveniles of Isotoma or related taxa (Potapov, (Cladonia sp.) in a cold temperate region (Matsumoto et 2001). Isotoma and Desoria are closely related, both belong- al., 2018). Desoria gracilliseta was absent during summer ing to the subfamily Isotominae (e.g., Greenslade and Pota- months. Juveniles of ca. 0.5 mm long were first observed in pov, 2015). Consequently, the coexistence of ud-Tetra and D. October and grew steadily during the winter to attain body gracilliseta, as well as their morphological similarities except lengths up to 2 mm by May. Subsequent surveys from mid- anal spines made us to hypothesize that ud-Tetra metamor- September to mid-October in the same site revealed the pres- phosed into juvenile D. gracilliseta. ence of a similar collembolan that possessed four anal spines. Metamorphosis is commonly known in Isotomidae and The collembola was tentatively assigned to Tetracanthura sp. Hypogastruridae. Fjellberg (1976) referred to the phenomenon (referred to as ud-Tetra), but differed from other known spe- of morphological change associated with seasonal change as cies belonging to the genus (Tanaka, 1982). Small numbers cyclomorphosis. Cyclomorphosis usually accompanies the of juvenile D. gracilliseta were present together with a large change in the furca and is considered an adaptation to facili- number of ud-Tetra in September. They grew larger and even- tate dispersal on the snow surface (Leinaas, 1981a, 1981b; tually outnumbered ud-Tetra with time. Hågvar, 1995; Sawahata, 2005). In the case of Hypogastrura 12 Naoyuki Matsumoto, Yasuhiko Suma, and Taizo Nakamori lapponica (Axelson, 1902), cyclomorphic change was not Large specimens, distinctive of D. gracilliseta collected associated with specific instars (Leinaas, 1981b). Epitoky on March 20, 2016, were used as reference since adult or includes morphological alterations in size and shape of anal large subadult specimens are required for taxonomic determi- spines, claws, mucro, and macrochaetae, and occurs during nations (Potapov, 2001). Specimens of ud-Tetra and juvenile sexual maturity (Fjellberg, 1977). Ecomorphosis occurs in and subadult stages of D. gracilliseta, also collected from the response to environmental factors such as rising temperatures same site, were used for DNA sequence analysis. DNA was and lowered soil moisture and includes the appearance of anal extracted from individual specimens according to Aoyama et spines (Hart, 1978). Our observations showed that ud-Tetra al. (2015). DNA sequences of partial regions of mitochondrial populations were replaced by D. gracilliseta populations but cytochorome c oxidase subunit 1 (CO1) and 16S ribosomal lacked the direct evidence to indicate metamorphosis such as RNA (16S rRNA) genes were determined as described by specimens undergoing ecdysis. Then, we conducted a detailed Potapov et al. (2010). Sequences were aligned using Muscle study of morphological development and population dynam- (Edgar, 2004; https://www.ebi.ac.uk/Tools/msa/muscle/) for ics throughout the growth cycle of the two types and applied both genes, and then cleaned using Gblocks 0.91b (Castresana DNA barcoding technique to confirm the identities of ud-Tetra 2000; http://molevol.cmima.csic.es/castresana/GBLOCKS_ and D. gracilliseta. server.html) for the 16S rRNA gene. To assess the associa- tion among ud-Tetra, juvenile and subadult individuals of D. Materials and methods gracilliseta, p-distances among specimens were estimated us- Sampling was made in two spots which were distant ing MEGA7 (Kumar et al., 2016) for both genes. Specimens ca. 100 m from each other and separated by a paved road in of Desoria trispinata (MacGillivray, 1896), congeneric with D. site B (Matsumoto et al., 2018). Site B had been cleared in gracilliseta, Tetracanthella sylvatica Yosii, 1939, a species of the late 1990’s and sparsely planted with Picea glehnii (Fr. spined genus, and Sahacanthella saoriae Nakamori & Pota- Schm.) Masters in Chitose, Hokkaido, northern Japan (42˚46’ pov, 2017, the species of spined genus used by Potapov et al. N, 141˚36’ E). A lichen, Cladonia sp., colonized the exposed (2017), were also included in the analysis (Table 1). The se- soil consisting predominately of pumice beneath the P. glehnii quences obtained were submitted to GenBank under accession canopy. Eight pieces of thallus of the lichen (7 × 7 cm) were numbers LC384348–LC384353 and LC384828–LC384833. sampled along with ca. 1 cm thick of pumice from each spot Specimens were deposited to the Tottori Prefectural Museum from September 16 to October 15 in 2017 to study population under accession numbers TRPM-AAr-0000756–TRPM- dynamics of ud-Tetra and juvenile D. gracilliseta. Collembo- AAr-0000761. lans were collected in 100% isopropanol with Tullgren fun- nels, sorted under a dissecting microscope, and preserved in Results 70% ethanol. The 2017 observations showed that numbers of ud-Tetra, Specimens of ud-Tetra and juvenile D. gracilliseta were first observed on September 16, peaked in late September, and arbitrarily sampled and mounted in Hoyer’s medium on glass dropped to zero by mid October (Table 2). Body lengths of slides for morphological comparison. Mounted specimens col- ud-Tetra remained consistent throughout the observation pe- lected from spots 1 and 2 in 2017 were photographed along riod, ranging from 0.47 to 0.5 mm (Table 2). Desoria gracil- with a ruler with a digital camera (Stylus SH-1, Olympus), liseta numbers were initially near zero but soon outnumbered and enlarged photos were used to determine body lengths as ud-Tetra in both spots (Table 2). Individuals of D. gracilliseta a distance between both ends of head and abdominal segment were initially larger than those of ud-Tetra and grew with time VI. Up to 20 individuals were determined. Antennal segment from 0.54 to 0.8 mm whereas the size of ud-Tetra remained IV and the furca of ud-Tetra and juvenile D. gracilliseta col- static throughout the sampling interval (Table 2). lected from spot 1 on October 4, 2016, were photographed us- Ud-Tetra possessed four anal spines on abdominal seg- ing an Olympus DP22 digital camera mounted on an Olympus ment VI, which was distinct from segment V (Fig. 1). When BX53 biological microscope. Lengths of antennal segment IV comparing individuals collected on the same date, ud-Tetra and the furca were precisely determined, using the DP2-SAL (Fig. 2A) closely resembled juvenile individuals of D. gracil- Olympus firmware. Number of individuals determined varied liseta (Fig. 2B) except that juveniles were slightly larger and from 11 to 20. lacked anal spines. Short spines were present on the head and Juvenile Desoria gracilliseta with anal spines 13 Table 1. Voucher sample information and GenBank accession number of sequence data used in molecular analysis. Voucher sample information GenBank accession number Reference Taxa Individual Museum ID COI gene 16S rRNA ID gene Desoria gracilliseta subadult ZI03 TRPM-AAr-0000760 LC384350 LC384830 Present study Desoria gracilliseta subadult ZI04 TRPM-AAr-0000761 LC384351 LC384831 Present study D. gracilliseta juvenile ZI05 TRPM-AAr-0000758 LC384352 LC384832 Present study D. gracilliseta juvenile ZI06 TRPM-AAr-0000758 LC384353 LC384833 Present study Tetracanthella sp. (ud-Tetra) ZI24 TRPM-AAr-0000756 LC384348 LC384828 Present study Tetracanthella