Morphology, phylogenetic position, and mating behaviour of a new Mesobiotus (Tardigrada) species from a rock pool in the Socorro Box Canyon (New Mexico, USA)

Figures & data

Figure 1. Type locality of Mesobiotus sp. nov, Freebird rock pool, Socorro Box Canyon located in near Socorro, New Mexico. Map was made using ArcGIS pro 3.1.1 (ESRI 2017) with Chihuahuan Desert boundary from Jornada Basin Spatial Data Laboratory (2006).

Table I. Sequences used for phylogenetic analysis.

Species	18S rRNA	28S rRNA	ITS-2	COI	Source
Mesobiotus huecoensis sp. nov.	OQ756249			OQ756246	This study
	OQ756248			OQ756247	This study
Mesobiotus diegoi Stec, 2022	OP142527	OP142520	OP142514	OP143858	Stec (2022)
	OP142526	OP142521	OP142515	OP143857	Stec (2022)
Mesobiotus maklowiczi Stec, 2022	OP142525	OP142518		OP143855	Stec (2022)
	OP142524	OP142519		OP143856	Stec (2022)
Mesobiotus peterseni (Maucci, 1991)	OP142528	OP142522	OP142516	OP143859	Stec (2022)
	OP142529	OP142523	OP142517	OP143860	Stec (2022)
Mesobiotus ethiopicus Stec & Kristensen, 2017	MF678793	MF678792	MN122776	MF678794	Stec and Kristensen (2017)
Mesobiotus datanlanicus Stec, 2019	MK584659	MK584658	MK584657	MK578905	Stec (2019)
Mesobiotus dilimanensis Itang et al., 2020	MN257048	MN257049	MN257050	MN257047	Itang et al. (2020)
Mesobiotus philippinicus Mapalo et al., 2016	KX129793	KX129794	KX129795	KX129796	Mapalo et al. (2016)
Mesobiotus insanis Mapalo et al., 2017	MF441488	MF441489	MF441490	MF441491	Mapalo et al. (2017)
Mesobiotus hilariae Vecchi et al., 2016	KT226070			KT226108	Vecchi et al. (2016)
Mesobiotus radiatus (Pilato etal., 1991)	MH197153	MH197152	MH197267	MH195147	Stec et al. (2018)
			MH197268	MH195148	Stec et al. (2018)
Mesobiotus romani Roszkowska et al., 2018	MH197158	MH197151	MH197150	MH195149	Roszkowska et al. (2018)
Mesobiotus harmsworthi (Murray, 1907)	MH197146	MH197264	MH197154	MH195150	Kaczmarek et al. (2018)
				MH195151	Kaczmarek et al. (2018)
Mesobiotus occultatus Kaczmarek et al., 2018	MH197147		MH197155	MH195152	Kaczmarek et al. (2018)
Mesobiotus furciger group species NO	MH197148	MH197265	MH197156	MH195153	Kaczmarek et al (2018)
Mesobiotus harmsworthi group species RU	MH197149	MH197266	MH197157	MH195154	Kaczmarek et al. (2018)
Mesobiotus fiedleri Kaczmarek et al., 2020	MH681585	MH681693	MH681724	MH676056	Kaczmarek et al. (2018)
Mesobiotus anastasiae Tumanov, 2020	MT903468	MT903612	MT903470	MT904513	Tumanov (2020)
Mesobiotus skorackii Kaczmarek et al., 2018		MW680636		MW656257	Kayastha et al. (2021)
Mesobiotus imperialis Stec, 2021	OL257854	OL257866		OL311514	Stec (2021)
	OL257855	OL257867		OL311515	Stec (2021)
Mesobiotus marmoreus Stec 2021	OL257856	OL257868	OL257861	OL311516	Stec (2021)
	OL257857	OL257869	OL257862	OL311517	Stec (2021)
	OL257858	OL257870	OL257863	OL311518	Stec (2021)
Mesobiotus cf. barabanovi	MN310392	MN310388	MN310390	MN313170	Kaczmarek et al. (2020)
Mesobiotus sp. Macro07_042	MW751942			MW727957	Short et al. (2022)
Mesobiotus cf. furciger Macro06_296	MW751936			MW727958	Short et al. (2022)
Mesobiotus cf. furciger Macro06_310	MW751937			MW727961	Short et al. (2022)
Mesobiotus cf. furciger Macro06_313	MW751939			MW727960	Short et al. (2022)
Mesobiotus cf. furciger CC_MF_4	MW751949			MW727933	Short et al. (2022)
Mesobiotus cf. furciger ABDC_MF_3	MW751944			MW727932	Short et al. (2022)
Mesobiotus cf. furciger KPRI_MF_1	MW751962			MW727934	Short et al. (2022)
Mesobiotus cf. furciger HMI_MF_1	MW751957			MW727941	Short et al. (2022)
Mesobiotus cf. furciger EBNI_MF_2	MW751952			MW727937	Short et al. (2022)
Mesobiotus cf. furciger EBNI_MF_4	MW751954			MW727938	Short et al. (2022)
Mesobiotus cf. furciger PSAI_MF_2	MW751967			MW727939	Short et al. (2022)
Mesobiotus cf. furciger Macro06_162	MW751934			MW727955	Short et al. (2022)
Mesobiotus cf. furciger Macro06_171	MW751935			MW727956	Short et al. (2022)
Mesobiotus cf. furciger JN07_MF_1	MW751959			MW727951	Short et al. (2022)
Mesobiotus cf. furciger JN07_MF_4	MW751960			MW727953	Short et al. (2022)
Mesobiotus cf. furciger JN07_MF_8	MW751961			MW727947	Short et al. (2022)
Mesobiotus cf. furciger FN01_MF_6	MW751955			MW727945	Short et al. (2022)
Macrobiotus kamilae Coughlan & Stec, 2019	MK737070	MK737064	MK737067	MK737920	Coughlan and Stec (2019)
				MK737921	Coughlan and Stec (2019)
Macrobiotus hannae Nowak & Stec, 2018	MH063922	MH063924	MH063923	MH057764	Nowak and Stec (2018)

Figure 2. Bayesian phylogenetic reconstruction of the genus Mesobiotus. Numbers above nodes indicate bayesian posterior probability (pp) (shown when pp = 1). Nodes with pp < 0.70 were collapsed. The new described species is highlighted in bold. Scale bar indicates the number of substitutions/site. Outrgoup taxa are not shown. For a complete version of the phylogenetic reconstruction including nodes with pp < 0.70 and outgroups see SM.03.

Figure 3. Mesobiotus huecoensis sp. nov. habitus and leg structures under PCM. (a) Holotype habitus. (b) Claws on leg I from paratype on slide SL1. (c) Claws on leg IV from holotype. (d) Granulation on external side of leg I from paratype on slide SL1. (e) Cuticular bulge on the internal side of leg III from paratype on slide SL1. (f) Granulation on legs IV from paratype on slide SL1. Arrowhead: continuous cuticular bar with with shadowed extensions toward the double muscle attachments. Arrow: flexible primary branch on claws IV. Indented arrowhead: horseshoe-shaped structure connects the anterior and posterior lunulae on claws IV. Empty arrowhead: granulation on legs. Empty indented arrowhead: cuticular bulge on internal leg surface. Images a- e were assembled from multiple focus stacks. Scalebars: 10 µm.

Figure 4. Mesobiotus huecoensis sp. nov. buccopharyngeal apparatus under PCM. (a) Buccopharyngeal apparatus of the holotype. (b) Dorsal view of the placoids row of the holotype. (c) Ventral view of the placoids row of the holotype. (d) Dorsal part of the oral cavity armature (OCA) of the holotype. (e) Dorsal part of the OCA of a paratype on slide SL1. (f) Ventral part of the OCA of the holotype. (g) Ventral part of the OCA of a paratype on slide SL1.Arrowhead: constriction in the third macroplacoid. Arrow: first (anterior) OCA band. Indented arrowhead: second (middle) OCA band. Empty indented arrowhead: third (posterior) OCA band. Empty arrowhead: mucrone behind the median part of the ventral third (posterior) OCA. Image a was assembled from a multiple focus stack. Scalebars: 10 µm.

Figure 5. Mesobiotus huecoensis sp. nov. eggs under PCM. (a) General view of the egg showing rounded processes and reticulation on the chorion. (b, c) Sections of egg processes. (d) Egg chorion covered by a reticulum. (e – j) Dorsal view of individual egg processes showing their variability. Arrowheads: pores on the processes. Image a was assembled from a multiple focus stack. Scalebars: (a) 10 µm, (b – j) 5 µm.

Figure 6. Mesobiotus huecoensis sp. nov. egg under SEM. (a) In toto view. (b) Egg processes showing pores on their lower half. (c) Focus on the reticulated eggshell chorion. Arrowheads: pores on the processes. Indented arrowhead: hole in the chorion reticulum. Scalebars:10 µm.

Table II. Measurements [in μm] and pt values of selected morphological structures of individuals of Mesobiotus huecoensis sp. nov.; specimens mounted in Hoyer’s medium.

CHARACTER	N		RANGE						MEAN		SD		Holotype
	µm	pt	µm			pt			µm	pt	µm	pt	µm	pt
Body length	29	24	257	–	682	877	–	1218	462	1080	128	94	604	1218
Buccopharyngeal tube
Buccal tube length	24		29.2	–	53.1		–		41.1	–	8.9	–	49.6	–
Stylet support insertion point	23	23	16.8	–	38.0	44.7	–	76.2	29.3	71.7	7.2	6.1	37.0	74.6
Buccal tube external width	24	24	3.7	–	32.4	11.4	–	61.0	6.4	15.0	5.7	9.8	7.1	14.3
Buccal tube internal width	24	24	2.2	–	5.3	7.5	–	10.0	3.5	8.4	0.9	0.6	4.1	8.3
Ventral lamina length	23	23	8.2	–	36.1	15.4	–	74.5	26.1	62.9	7.4	11.1	31.5	63.5
Placoid lengths
Macroplacoid 1	24	24	2.4	–	7.3	8.2	–	14.1	4.8	11.3	1.8	2.1	7.0	14.1
Macroplacoid 2	24	24	2.3	–	6.0	7.8	–	12.3	4.2	10.1	1.3	1.2	5.9	11.9
Macroplacoid 3	24	24	2.6	–	7.3	8.8	–	13.7	4.7	11.2	1.6	1.5	6.2	12.5
Microplacoid	24	24	1.3	–	4.9	4.3	–	9.4	2.8	6.6	1.1	1.5	4.0	8.1
Macroplacoid row	24	24	9.0	–	22.3	30.7	–	42.0	15.5	37.1	4.6	3.5	20.7	41.7
Placoid row	24	24	11.4	–	28.2	38.9	–	53.1	19.2	45.8	5.8	4.4	25.1	50.6
Claw I heights
External base	23	20	3.6	–	10.5	12.3	–	20.5	6.4	15.2	2.1	2.3	6.5	13.1
External primary branch	29	24	9.1	–	19.5	27.8	–	38.0	13.7	32.8	3.3	2.4	16.6	33.5
External secondary branch	28	24	6.1	–	13.8	20.0	–	27.0	10.3	24.9	2.5	2.0	13.1	26.4
External base/primary branch (cct)	23		35.5	–	63.5		–		46.0	–	6.8	–	39.2	–
Internal base	23	20	3.3	–	10.0	10.9	–	19.5	6.0	14.1	2.1	2.5	6.7	13.5
Internal primary branch	29	24	8.1	–	18.6	23.1	–	36.3	13.2	31.2	3.3	2.6	16.0	32.3
Internal secondary branch	29	24	6.1	–	14.2	17.9	–	27.7	9.6	23.0	2.5	2.4	12.9	26.0
Internal base/primary branch (cct)	23		33.8	–	62.0		–		45.3	–	7.7	–	41.9	–
Claw II heights
External base	24	20	3.3	–	9.9	11.3	–	19.0	6.6	15.8	1.9	2.3	8.4	16.9
External primary branch	28	23	9.5	–	21.1	31.9	–	40.4	14.9	35.5	3.4	2.5	19.1	38.5
External secondary branch	28	23	6.3	–	16.5	19.5	–	32.2	10.7	25.4	2.9	2.7	14.0	28.2
External base/primary branch (cct)	24		32.7	–	54.0		–		43.6	–	5.6	–	44.0	–
Internal base	25	21	3.5	–	9.9	9.6	–	19.3	6.4	14.7	2.0	2.4	7.1	14.3
Internal primary branch	28	23	9.4	–	19.9	30.2	–	38.0	14.4	33.9	3.6	2.1	16.4	33.1
Internal secondary branch	28	23	6.6	–	16.0	22.3	–	31.3	10.9	25.8	3.0	2.3	14.1	28.4
Internal base/primary branch (cct)	25		29.5	–	60.5		–		44.1	–	7.3	–	43.3	–
Claw III heights
External base	23	20	3.7	–	10.6	9.9	–	20.7	7.1	16.5	2.1	2.9	7.8	15.7
External primary branch	28	23	10.0	–	20.8	32.5	–	42.2	15.4	36.5	3.6	2.4	17.5	35.3
External secondary branch	28	23	5.9	–	41.2	19.5	–	80.8	12.2	28.7	6.4	11.7	14.1	28.4
External base/primary branch (cct)	23		30.6	–	58.4		–		44.6	–	8.0	–	44.6	–
Internal base	20	18	3.8	–	10.8	10.5	–	21.6	6.8	15.8	2.3	3.0	7.9	15.9
Internal primary branch	28	23	8.9	–	21.4	28.0	–	41.7	14.5	34.5	3.9	3.1	17.3	34.9
Internal secondary branch	27	22	6.6	–	16.3	20.3	–	30.7	10.5	25.0	3.0	2.5	13.9	28.0
Internal base/primary branch (cct)	20		31.6	–	58.4		–		46.3	–	7.1	–	45.7	–
Claw IV heights
Anterior base	24	20	3.7	–	11.8	12.2	–	23.6	7.2	16.8	2.6	3.4	8.8	17.7
Anterior primary branch	28	23	9.0	–	22.9	30.8	–	48.4	17.0	40.3	4.4	4.1	18.5	37.3
Anterior secondary branch	28	23	6.9	–	16.6	21.5	–	32.4	11.6	27.7	3.3	2.8	15.4	31.0
Anterior base/primary branch (cct)	24		29.7	–	51.5		–		41.3	–	6.8	–	47.6	–
Posterior base	24	19	3.9	–	11.0	10.3	–	20.7	7.0	16.2	2.2	2.8	9.8	19.8
Posterior primary branch	28	23	11.3	–	24.8	37.9	–	49.9	17.8	42.5	4.1	3.0	19.8	39.9
Posterior secondary branch	28	23	6.6	–	16.6	22.3	–	31.3	11.8	28.0	3.0	2.5	14.8	29.8
Posterior base/primary branch (cct)	24		24.6	–	49.5		–		38.5	–	6.0	–	49.5	–

Table III. Measurements [in μm] of the eggs of Mesobiotus huecoensis sp. nov.; eggs mounted in Hoyer’s medium; process base/height ratio is expressed as percentage.

CHARACTER	N	RANGE			MEAN	SD
Egg bare diameter	15	60.9	–	78.6	70.8	5.1
Egg full diameter	15	74.6	–	92.3	85.9	5.2
Process height	48	2.3	–	6.4	4.8	0.8
Process base width	48	4.6	–	7.5	6.1	0.7
Process base/height ratio	48	93%	–	250%	131%	32%
Inter-process distance	45	0.7	–	4.7	2.6	0.9
Number of processes on the egg circumference	15	21	–	30	26.5	2.8

Table IV. Measurements [in μm] of the sperms of Mesobiotus huecoensis sp. nov. stained with Phalloidin-TRITC and Hoechst 33,342.

CHARACTER	N	RANGE			MEAN	SD
Acrosome	8	4.1	–	6.2	5.0	0.8
Nucleus	13	14.4	–	16.7	15.5	0.6
Midpiece	13	8.6	–	10.9	9.5	0.7
Tail	10	20.6	–	24.5	22.7	1.2
Total length	7	51.5	–	53.5	52.7	0.6

Figure 7. Mesobiotus huecoensis sp. nov. male gonad and spermatozoa. (a) Male gonad under PCM stained with orcein showing different maturation stages of the male gamets. (b) Stained spermatozoa under Confocal Microscopy, blue represents stained DNA (nucleus), whereas white represents Phalloidin-stained actin (acrosome, midpiece, tail). Black arrowhead: spermatids. Black indented arrowhead: mature spermatozoa. White arrowhead: acrosome. White indented arrowhead: midpiece. White arrow: tail. Scalebars:10 µm.

Figure 8. Comparative morphology of Mesobiotus montanus morphogroup species under PCM. (a) M. lusitanicus from type series. (b) M. montanus. (c) M. mottai from type series. (d) M. peterseni. Scale bars:10 µm.

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Data availability statement

The data that support the findings of this study are openly available in Figshare at DOI:10.6084/m9.figshare.22656586 under CC-BY 4.0 license and in GenBank (see Table 1 for accession numbers).