Research Article |
Corresponding author: Andrzej Lesicki ( alesicki@amu.edu.pl ) Academic editor: Martin Haase
© 2024 Joanna R. Pieńkowska, Giuseppe Manganelli, Małgorzata Proćków, Debora Barbato, Katarzyna Sosnowska, Folco Giusti, Andrzej Lesicki.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Pieńkowska JR, Manganelli G, Proćków M, Barbato D, Sosnowska K, Giusti F, Lesicki A (2024) Next step in Monacha cantiana (Montagu, 1803) phylogeography: northern French and Dutch populations (Eupulmonata, Stylommatophora, Hygromiidae). ZooKeys 1198: 55-86. https://doi.org/10.3897/zookeys.1198.119738
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Features of shell and genitalia as well as nucleotide sequences of selected mitochondrial and nuclear genes of specimens of Monacha cantiana from ten northern French and two Dutch populations were compared with the same features of British and Italian populations. They were found to be very similar to populations previously identified as belonging to the CAN-1 lineage of M. cantiana. This confirms previous suggestions that M. cantiana was introduced to western Europe (England, France and the Netherlands) in historical times.
16SrDNA, COI, genitalia, H3, ITS2, mitochondrial and nuclear genes, nucleotide sequences, population distribution, shell
Monacha Fitzinger, 1833 is a species-rich genus including numerous nominal species diversified mainly in the Anatolian and European parts of Turkey, in the southern parts of the Balkans and in Italy (
Monacha cantiana, commonly known as the Kentish snail, was described by
It has been suggested that this species was introduced to the British Isles in historical times (
Monacha cantiana has been reported from France (
The aim of the present research was: 1) to study morphological (shell and genitalia) and molecular variation in specimens of M. cantiana collected in northern France and the Netherlands in order to clarify their relations to the British and Italian populations; 2) to test the hypothesis that the English, French and Dutch populations originated from the same introduced propagules.
Specimens from ten French and two Dutch populations of Monacha cantiana were considered for analysis of the variability of their molecular and morphological (shell and genitalia) features (Table
List of localities of Monacha cantiana s.l. populations used for molecular and morphological (SH shell, AN genitalia) research.
Localities | Current taxonomy | Clade | Designation of DNA voucher sps | COI | Long 16SrDNA | H3 | 5.8SrDNA + ITS2 + 28SrDNA | PCA and RDA | Figs | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
No. | coordinates | country and site | collector / date / no. of specimens (collection) | new haplotype | GenBank ## | new haplotype | GenBank ## | new haplotype | GenBank ## | new haplotype | GenBank ## | |||||
1 | 50°47'56.7"N, 02°00'57.5"E | France, Pas-de-Calais, Bonningues-lès-Ardres, vegetation under shrubs, 42 m a.s.l. | M. Proćków / 20.06.2018 / 5 (MNHW* F.18.13) | M. cantiana | CAN-1 | Ard1 | 16S 1 | OR918363 | H3 1 | OR939858 | ITS2 1 | OR917347 | AN | |||
Ard2 | COI 1 | OR918493 | 16S 1 | OR918364 | H3 2 | OR939859 | ITS2 2 | OR917348 | AN | |||||||
Ard3 | 16S 1 | OR918365 | H3 1 | OR939860 | ITS2 1 | OR917349 | ||||||||||
Ard4 | COI 1 | OR918494 | 16S 2 | OR918366 | H3 1 | OR939861 | ITS2 1 | OR917350 | ||||||||
Ard5 | H3 3 | OR939862 | ||||||||||||||
2 | 50°49'28.1"N, 01°44'01.9"E | France, Pas-de-Calais, Blecquenecques n. Marquise, roadside, 26 m a.s.l. | M. Proćków / 20.06.2018 / 5 (MNHW F.18.10) | M. cantiana | CAN-1 | Ble1 | COI 1 | OR918495 | 16S 3 | OR918367 | H3 1 | OR939863 | SH/AN | SH/AN | ||
Ble2 | COI 1 | OR918496 | 16S 1 | OR918368 | H3 1 | OR939864 | ITS2 3 | OR917351 | ||||||||
Ble4 | COI 1 | OR918497 | 16S 3 | OR918369 | H3 1 | OR939865 | ||||||||||
Ble5 | COI 1 | OR918498 | H3 1 | OR939866 | ITS2 4 | OR917352 | ||||||||||
3 | 50°40'56.7"N, 02°03'39.1"E | France, Pas-de-Calais, Larré, vegetation along stream, 65 m a.s.l. | M. Proćków / 20.06.2018 / 5 (MNHW F.18.14) | M. cantiana | CAN-1 | Lar1 | COI 1 | OR918499 | 16S 3 | OR918370 | H3 3 | OR939867 | ITS2 5 | OR917353 | ||
Lar2 | COI 2 | OR918500 | 16S 4 | OR918371 | H3 1 | OR939868 | ITS2 6 | OR917354 | ||||||||
Lar3 | COI 1 | OR918501 | 16S 4 | OR918372 | H3 1 | OR939869 | ITS2 1 | OR917355 | ||||||||
Lar4 | COI 3 | OR918502 | H3 4 | OR939870 | ITS2 7 | OR917356 | ||||||||||
Lar5 | COI 1 | OR918503 | 16S 4 | OR918373 | H3 1 | OR939871 | ITS2 7 | OR917357 | ||||||||
4 | 50°47'48.2"N, 01°56'34.4"E | France, Pas-de-Calais, Licques, vegetation along road, 81 m a.s.l. | M. Proćków / 20.06.2018 / 5 (MNHW F.18.12) | M. cantiana | CAN-1 | Lic1 | COI 1 | OR918504 | 16S 1 | OR918374 | H3 1 | OR939872 | ITS2 1 | OR917358 | ||
Lic2 | COI 1 | OR918505 | 16S 3 | OR918375 | H3 1 | OR939873 | ITS2 8 | OR917359 | ||||||||
Lic3 | 16S 5 | OR918376 | H3 3 | OR939874 | ITS2 1 | OR917360 | ||||||||||
Lic4 | COI 1 | OR918506 | 16S 5 | OR918377 | H3 1 | OR939875 | ITS2 9 | OR917361 | ||||||||
Lic5 | COI 1 | OR918507 | 16S 1 | OR918378 | H3 1 | OR939876 | ITS2 1 | OR917362 | ||||||||
5 | 49°54'23.6"N, 01°30'58.9"E | France, Seine-Maritime, Béthencourt n. Grandcourt, vegetation under trees, 97 m a.s.l. | M. Proćków / 23.06.2018 / 5 (MNHW F.18.22) | M. cantiana | CAN-1 | Bet1 | COI 1 | OR918508 | 16S 3 | OR918379 | H3 1 | OR939877 | ITS2 10 | OR917363 | SH/AN | SH/AN |
Bet2 | COI 1 | OR918509 | H3 1 | OR939878 | ITS2 11 | OR917364 | ||||||||||
Bet3 | 16S 6 | OR918380 | H3 1 | OR939879 | ||||||||||||
Bet4 | COI 1 | OR918510 | 16S 6 | OR918381 | H3 1 | OR939880 | ITS2 12 | OR917365 | ||||||||
Bet5 | COI 1 | OR918511 | 16S 3 | OR918382 | H3 1 | OR939881 | ITS2 13 | OR917366 | ||||||||
6 | 49°55'05.6"N, 01°31'38.1"E | France, Seine-Maritime, Pierrepont, forest edge, 146 m a.s.l. | M. Proćków / 23.06.2018 / 5 (MNHW F.18.21) | M. cantiana | CAN-1 | Pie1 | COI 1 | OR918512 | 16S 3 | OR918383 | H3 1 | OR939882 | ITS2 14 | OR917367 | SH/AN | SH/AN |
Pie2 | COI 1 | OR918513 | H3 1 | OR939883 | ITS2 1 | OR917368 | ||||||||||
Pie3 | COI 1 | OR918514 | H3 1 | OR939884 | ||||||||||||
Pie4 | COI 1 | OR918515 | 16S 3 | OR918384 | H3 1 | OR939885 | ITS2 15 | OR917369 | ||||||||
7 | 50°04'05.1"N, 01°52'20.9"E | France, Somme, Épagne-Épagnette, roadside, 13 m a.s.l. | M. Proćków / 19.06.2018 / 5 (MNHW F.18.08) | M. cantiana | CAN-1 | Epa1 | COI 1 | OR918516 | 16S 7 | OR918385 | H3 3 | OR939886 | ITS2 16 | OR917370 | SH/AN | AN |
Epa2 | COI 1 | OR918517 | 16S 3 | OR918386 | H3 1 | OR939887 | ITS2 1 | OR917371 | ||||||||
Epa3 | COI 4 | OR918518 | H3 1 | OR939888 | ITS2 17 | OR917372 | ||||||||||
Epa4 | 16S 8 | OR918387 | H3 5 | OR939889 | ITS2 18 | OR917373 | ||||||||||
Epa5 | COI 1 | OR918519 | 16S 9 | OR918388 | H3 1 | OR939890 | ||||||||||
8 | 50°16'54.7"N, 01°37'41.9"E | France, Somme, Froise, forest edge, 86 m a.s.l. | M. Proćków / 19.06.2018 / 5 (MNHW F.18.20) | M. cantiana | CAN-1 | Fro1 | 16S 4 | OR918389 | H3 2 | OR939891 | ||||||
Fro2 | COI 1 | OR918520 | 16S 10 | OR918390 | H3 1 | OR939892 | ITS2 19 | OR917374 | ||||||||
Fro3 | COI 1 | OR918521 | 16S 11 | OR918391 | H3 1 | OR939893 | ITS2 1 | OR917375 | ||||||||
Fro4 | COI 1 | OR918522 | 16S 12 | OR918392 | H3 1 | OR939894 | ||||||||||
Fro5 | COI 1 | OR918523 | 16S 13 | OR918393 | H3 2 | OR939895 | ||||||||||
9 | 49°44'14.7"N, 01°47'53.9"E | France, Oise, Escales-Saint-Pierre, roadside, 164 m a.s.l. | M. Proćków / 19.06.2018 / 5 (MNHW F.18.06) | M. cantiana | CAN-1 | Esc1 | COI 1 | OR918524 | 16S 14 | OR918394 | H3 1 | OR939896 | ITS2 20 | OR917376 | SH/AN | SH/AN |
Esc2 | COI 1 | OR918525 | H3 2 | OR939897 | ITS2 21 | OR917377 | ||||||||||
Esc3 | COI 1 | OR918526 | 16S 14 | OR918395 | H3 6 | OR939898 | ITS2 22 | OR917378 | ||||||||
Esc4 | COI 1 | OR918527 | H3 1 | OR939899 | ITS2 23 | OR917379 | ||||||||||
Esc5 | COI 1 | OR918528 | 16S 15 | OR918396 | H3 6 | OR939900 | ITS2 17 | OR917380 | ||||||||
10 | 49°27'38.2"N, 02°03'35.0"E | France, Oise, Fouquenies, vegetation along forest road, 29 m a.s.l. | M. Proćków / 19.06.2018 / 5 (MNHW F.18.05) | M. cantiana | CAN-1 | Fou1 | COI 1 | OR918529 | 16S 3 | OR918397 | H3 3 | OR939901 | ITS2 24 | OR917381 | ||
Fou2 | COI 5 | OR918530 | 16S 16 | OR918398 | H3 1 | OR939902 | ITS2 1 | OR917382 | ||||||||
Fou3 | 16S 3 | OR918399 | H3 7 | OR939903 | ||||||||||||
Fou4 | 16S 17 | OR918400 | H3 7 | OR939904 | ITS2 25 | OR917383 | ||||||||||
Fou5 | COI 1 | OR918531 | 16S 18 | OR918401 | H3 1 | OR939905 | ITS2 26 | OR917384 | ||||||||
11 | 51°32'57.0"N,, 03°39'27.9"E | The Netherlands, Veere, edge of forest, 15 m a.s.l. | M. Proćków / 6.06.2019/ 5 (MNHW NL.19.02) | M. cantiana | CAN-1 | Vee1-1 | COI 1 | OR918532 | 16S 19 | OR918402 | H3 1 | OR939906 | ||||
Vee1-2 | COI 1 | OR918533 | 16S 1 | OR918403 | H3 8 | OR939907 | ||||||||||
Vee1-3 | COI 1 | OR918534 | 16S 3 | OR918404 | H3 1 | OR939908 | ||||||||||
Vee1-4 | COI 1 | OR918535 | 16S 19 | OR918405 | H3 1 | OR939909 | ||||||||||
Vee1-5 | COI 6 | OR918536 | 16S 3 | OR918406 | H3 1 | OR939910 | ||||||||||
12 | 51°32'57.1"N,, 03°39'40.1"E | The Netherlands, Veere 6, vegetation near windmill, 81 m a.s.l. | M. Proćków / 7.06.2019/ 5 (MNHW NL.19.07) | M. cantiana | CAN-1 | Vee2-1 | 16S 3 | OR918407 | H3 1 | OR939911 | ||||||
Vee2-2 | COI 7 | OR918537 | 16S 3 | OR918408 | H3 5 | OR939912 | ||||||||||
Vee2-3 | COI 1 | OR918538 | 16S 19 | OR918409 | H3 1 | OR939913 | ||||||||||
Vee2-4 | COI 1 | OR918539 | 16S 3 | OR918410 | H3 1 | OR939914 | ||||||||||
Vee2-5 | OR918540 | 16S 3 | OR918411 | H3 1 | OR939915 | |||||||||||
13. | 50°46'23.5"N, 01°50'06.3"W | United Kingdom, Hurn, vegetation along road, 7 m a.s.l. | M. Proćków / 15.06.2022/ 2 (MNHW GB.22.04) | M. cantiana | CAN-1 | Hum1 | 16S 1 | OR918412 | H3 1 | OR939916 | ITS2 1 | OR917385 | ||||
Hum2 | COI 8 | OR918541 | H3 9 | OR939917 | ||||||||||||
14. | 51°17'43.7"N, 01°29'34.9"W | United Kingdom, Vernhams Dean, vegetation along shaded path, 136 m a.s.l. | M. Proćków / 15.06.2022/ 4 (MNHW GB.22.05) | M. cantiana | CAN-1 | Ver1 | 16S 3 | OR918413 | H3 9 | OR939918 | ITS2 1 | OR917386 | ||||
Ver2 | 16S 3 | OR918414 | H3 9 | OR939919 | ITS2 1 | OR917387 | ||||||||||
Ver3 | H3 10 | OR939920 | ITS2 27 | OR917388 | ||||||||||||
Ver4 | 16S 3 | OR918415 | H3 1 | OR939921 | ITS2 1 | OR917389 | ||||||||||
15. | 51°17'32.3"N, 01°29'10.9"W | United Kingdom, Upton, vegetation along road, 120 m a.s.l. | M. Proćków / 15.06.2022/ 2 (MNHW GB.22.06) | M. cantiana | CAN-1 | Upt1 | 16S 3 | OR918416 | H3 1 | OR939922 | ITS2 1 | OR917390 | ||||
Upt2 | 16S 3 | OR918417 | H3 1 | OR939923 | ITS2 1 | OR917391 | ||||||||||
16. | 55°02'13.6"N, 01°42'51.0"W | United Kingdom, Newcastle upon Tyne, vegetation near airport, 80 m a.s.l. | M. Proćków / 15.06.2022/ 6 (MNHW GB.22.07) | M. cantiana | CAN-1 | New1 | COI 9 | OR918542 | 16S 20 | OR918418 | H3 9 | OR939924 | ITS2 1 | OR917392 | ||
New2 | 16S 20 | OR918419 | H3 9 | OR939925 | ITS2 1 | OR917393 | ||||||||||
New3 | COI 10 | OR918543 | 16S 20 | OR918420 | H3 9 | OR939926 | ITS2 1 | OR917394 | ||||||||
New4 | COI 9 | OR918544 | 16S 20 | OR918421 | H3 1 | OR939927 | ITS2 1 | OR917395 | ||||||||
New5 | COI 1 | OR918545 | 16S 3 | OR918422 | H3 9 | OR939928 | ITS2 1 | OR917396 | ||||||||
New6 | COI 9 | OR918546 | 16S 20 | OR918423 | H3 1 | OR939929 | ITS2 1 | OR917397 | ||||||||
17. | 53°31'29"N, 01°27'54"W | United Kingdom, Barrow near Barnsley | R.A.D. Cameron / 10.2011 / 5 (FGC* 40329) | M. cantiana | CAN-1 | 8FG-1 | MG208884 | 16S 1 | OR918424 | MG209031 | ITS2 1 | OR917398 | ||||
8FG-2 | MG208885 | 16S 1 | OR918425 | MG209032 | ITS2 1 | OR917399 | ||||||||||
18. | 53°25'04.2"N, 01°24'00.5"W | United Kingdom, Rotherham | R.A.D. Cameron / 07.2015 / 7 (DCBC*) | M. cantiana | CAN-1 | Sit1-1 | MG208893 | 16S 1 | OR918426 | MG209035 | ITS2 28 | OR917400 | ||||
19. | 53°24'49.1"N, 01°24'36.6"W | United Kingdom, Sheffield | R.A.D. Cameron / 07.2015 / 6 (DCBC) | M. cantiana | CAN-1 | Sit2-1 | MG208899 | 16S 21 | OR918427 | MG209038 | ITS2 1 | OR917401 | ||||
20 | 42°28'41.05"N, 13°05'09.46"E | Italy, Latium, Gole del Velino, near Sigillo (Posta, Rieti) | A. Hallgass / 30.09.2012 / 8 (FGC 42960) | M. cantiana | CAN-1 | 4FG-1 | MG208905 | 16S 24 | OR918428 | MG209039 | ITS2 29 | OR917402 | ||||
4FG-2 | MG208910 | 16S 25 | OR918429 | MG209042 | ITS2 29 | OR917403 | ||||||||||
21. | 42°43'39.87"N, 13°16'01.44"E | Italy, Latium, Valle del Tronto (Accumoli, Rieti) | A. Hallgass / 30.09.2012 / 4 (FGC 42963) | M. cantiana | CAN-1 | Tro1 | MG208921 | 16S 26 | OR918430 | MG209043 | ITS2 1 | OR917404 | ||||
22. | 42°07'53.39"N, 13°01'39.81"E | Italy, Latium, Valle del Turano, near Turania (Rieti) | A. Hallgass / 04.11.2013 / 2 (FGC 42969) | M. cantiana | CAN-1 | Tur5-1 | MG208923 | 16S 27 | OR918431 | MG209048 | ITS2 29 | OR917405 | ||||
Tur5-2 | MG208924 | 16S 28 | OR918432 | |||||||||||||
23. | 43°44'26.18"N, 12°17'13.71"E | Italy, Tuscany, Sasso di Simone, Rifugio Casa del Re (Sestino, Arezzo) | G. Manganelli / 21.10.2017 / 4 (FGC 47484) | M. cantiana | CAN-2 | Sim-1 | COI 11 | OR918547 | 16S 22 | OR918433 | H3 1 | OR939930 | ||||
Sim-2 | COI 11 | OR918548 | 16S 23 | OR918434 | H3 1 | OR939931 | ||||||||||
24. | 45°11'59.85"N, 10°58'49.30"E | Italy, Venetum, Sorgà (Verona) | A. Hallgass / 09.2012 / 6 (FGC 42964) | M. cantiana | CAN-2 | 12FG-1 | MG208925 | 16S 29 | OR918435 | MG209050 | ITS2 30 | OR917406 | ||||
12FG-2 | MG208928 | 16S 30 | OR918436 | H3 1 | OR939932 | ITS2 31 | OR917407 | |||||||||
25. | 48°15'25.50"N, 16°30'46.38"E | Austria, Breitenlee, abandoned railway station | M. Duda / 09.2015 / 3 (FGC 44020) | M. cantiana | CAN-3 | Dud-2 | MG208938 | 16S 31 | OR918437 | MG209056 | ITS2 32 | OR917408 | ||||
26. | 43°46'11.79"N, 07°22'21.50"E | France, Alpes-Maritimes, Vallée de Peillon, Sainte Thècle | A. Hallgass / 24.10.2011/ 5 (FGC 40320) | M. cemenelea | CAN-4 | 3FG-1 | MG208939 | 16S 32 | OR918438 | MG209058 | ITS2 33 | OR917409 | ||||
3FG-2 | MG208940 | 16S 32 | OR918439 | MG209059 | ITS2 34 | OR917410 |
The material examined originated from the populations listed in Table
Sixty-six specimens of the six lineages of M. cantiana s.l. (CAN-1, CAN-2, CAN-3, CAN-4, CAN-5, and CAN-6) (
AH aperture height,
AW aperture width,
LWfW last whorl final width,
LWmW last whorl medial width,
LWaH height of adapical sector of last whorl,
LWmH height of medial sector of last whorl,
PWH penultimate whorl height,
PWfW penultimate whorl final width,
PWmW penultimate whorl medial width,
SD shell diameter,
SH shell height,
UD umbilicus diameter.
Sixty-four specimens of the six lineages of M. cantiana s.l. (CAN-1, CAN-2, CAN-3, CAN-4, CAN-5 and CAN-6) (
BC bursa copulatrix,
BW body wall,
DBC duct of bursa copulatrix,
DG digitiform glands,
E epiphallus (from base of flagellum to beginning of penial sheath),
F flagellum,
FO free oviduct,
GA genital atrium,
GAR genital atrium retractor,
P penis,
PP penial papilla,
SOD spermoviduct,
V vagina,
VA vaginal appendix (also known as appendicula),
VAS vaginal appendix sac,
VD vas deferens.
Six anatomical variables (DBC, E, F, P, V, VA) were measured using a calliper under a light microscope (0.01 mm) (
Detailed methods of multivariate ordination by Principal Component Analysis (PCA) and Redundancy Analysis (RDA), performed on the original shell and genitalia matrices as well as on the Z-matrices (shape-related matrices), are described in our previous papers (
We used 95% confidence interval ellipses to evaluate the uncertainty of the estimate of the population mean (centroid) of the data sample. The function ordiellipse with standard errors in the package vegan (
Eighty-eight specimens representing 26 populations of the four lineages of M. cantiana s.l. (CAN-1, CAN-2, CAN-3, and CAN-4;
Two mitochondrial and two nuclear gene fragments were analysed, namely cytochrome c oxidase subunit 1 (COI), 16S ribosomal DNA (16SrDNA), histone 3 (H3) and an internal transcribed spacer 2 of rDNA (ITS2) flanked by the 3’end of 5.8SrDNA and the 5’end of 28SrDNA, respectively. Sequences were edited by eye using BioEdit, v. 7.0.6 (
Estimates of genetic distances between the COI sequences obtained in this study and other sequences from GenBank were conducted with MEGA7 using the Kimura two-parameter model (K2P) (
To infer the phylogenetic relationships the following programmes were used: MEGA7 (
For each alignment file, best nucleotide substitution models were specified according to the Bayesian Information Criterion (BIC) (see captions to figures). Phylogenetic analyses performed with IQ-Tree, RAxML and MrBayes for three sets of concatenated sequences were done dividing the data set into 2 or 4 partitions: (1) COI, (2) 16SrDNA or (1) COI, (2) 16SrDNA, (3) H3, (4) 5.8SrDNA + ITS2 + 28SrDNA. Best substitution models were inferred according to the Bayesian Information Criterion (BIC) for each of the partitions by MODELFINDER (
The robustness of the NJ and ML trees generated by MEGA7 were assessed by bootstrap analysis with 1000 replicates (
Shells of French specimens of M. cantiana (Fig.
RDA with French specimens and “lineage” constraint on the shape and size matrix (Fig.
Analysis of French specimens with “lineage” constraint on the original matrix (A–C) and Z-matrix (shape-related) (D–F) of selected shell sections. Principal component analysis (PCA) (A, D) and redundancy analysis (RDA) with groups shown as ellipses representing 95% confidence intervals with standard errors (B, E) and as convex hull polygons (C, F).
RDA on the shape (Z) matrix (Fig.
French specimens of M. cantiana have distal genitalia (Figs
Distal genitalia of Monacha cantiana from France. Specimen Bet1 from Seine-Maritime, Béthencourt n. Grandcourt (A–C) and specimen Ble1 from Pas-de-Calais, Blecquenecques n. Marquise (D–F). Distal genitalia (A, D), transverse sections of medial epiphallus (B, E) and apical penial papilla (C, F).
Distal genitalia genitalia of Monacha cantiana from France. Specimen Esc1 from Oise, Escales-Saint-Pierre (A–C) and specimen Epa1 from Somme, Épagne-Épagnette, roadside (D). Distal genitalia (A), transverse sections of medial epiphallus (B), apical penial papilla (C) and internal structure of distal genitalia (D).
RDA with French specimens and “lineage” constraint on the shape and size matrix (Fig.
Analysis of French specimens with “lineage” constraint on the original matrix (A–C) and Z-matrix (shape-related) (D–F) of selected genital sections. Principal component analysis (PCA) (A, D) and redundancy analysis (RDA) with groups shown as ellipses representing 95% confidence intervals with standard errors (B, E) and as convex hull polygons (C, F).
RDA on the shape (Z) matrix (Figs
Although sequences of all the genes analysed (COI, 16SrDNA, H3, and ITS2 with 5.8SrDNA and 28SrDNA) were not obtained from all 88 specimens (Table
The phylogenetic analysis of COI sequences obtained from the specimens and comparative sequences derived from GenBank is shown in Fig.
Maximum Likelihood (ML) tree of COI haplotypes of Monacha cantiana. New COI sequences of M. cantiana (Table
K2P genetic distances (Table
K2P genetic distances between COI sequences of the populations analysed.
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | ||
---|---|---|---|---|---|---|---|---|---|
M. cantiana CAN-1 of French populations | 1 | 1.1 | |||||||
M. cantiana CAN-1 of Dutch populations | 2 | 0.7 | 0.2 | ||||||
M. cantiana CAN-1 of English populations | 3 | 0.9 | 0.5 | 0.7 | |||||
M. cantiana CAN-1 of Italian populations | 4 | 1.2 | 0.8 | 0.9 | 0.6 | ||||
M. cantiana CAN-2 of Italian populations | 5 | 4.1 | 3.7 | 3.8 | 3.5 | 2.4 | |||
M. cantiana s.l. CAN-3 of Italian populations | 6 | 18.7 | 18.6 | 18.6 | 18.5 | 18.3 | 1.0 | ||
M. cantiana s.l. CAN-3 of Austrian populations | 7 | 18.8 | 18.7 | 18.7 | 18.7 | 18.5 | 1.5 | 1.0 | |
M. cantiana s.l. CAN-4 (M. cemenelea) of French populations | 8 | 18.3 | 18.2 | 18.1 | 18.0 | 18.6 | 5.6 | 6.1 | 0.9 |
Results similar to those of COI analysis were obtained for other single gene analyses (Suppl. materials
The phylogenetic tree for concatenated sequences were similar in ML analyses obtained with different software. The tree for mitochondrial gene sequences (COI+16SrDNA) in Fig.
Maximum Likelihood (ML) tree of concatenated COI and 16SrDNA haplotypes of Monacha cantiana. New COI and 16SrDNA sequences of M. cantiana (Table
Maximum Likelihood (ML) tree of concatenated H3 and ITS2 (flanked with 5.8S and 28SrDNA) haplotypes of Monacha cantiana. New H3 and ITS2 sequences of M. cantiana (Table
Maximum Likelihood (ML) tree of concatenated COI, 16SrDNA, H3, and ITS2 (flanked with 5.8S and 28SrDNA) haplotypes of Monacha cantiana. COI, 16SrDNA, H3, and ITS2 sequences of M. cantiana were compared with sequences of M. cantiana s.l. and M. cartusiana obtained from GenBank (Suppl. materials
At a first glance, the shells and genitalia of the French specimens do not differ from those of the other populations assigned to CAN-1, which in turn are similar to those of the populations of the CAN-2, CAN-3 and CAN-4 lineages (see
The results of molecular analysis were consistent with those of morphological analysis (shell and genital structure). Both showed that the populations from northern France should be assigned to the CAN-1 lineage. In this sense, the molecular results complement the conclusions of
Prior suggestions that M. cantiana was introduced into England in historical times (
We thank Jarosław Bogucki (Poznań, Poland) for drawing the map (Fig.
The authors have declared that no competing interests exist.
No ethical statement was reported.
DB and GM were funded under the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.4 – Call for tender No. 3138 of 16 December 2021, rectified by Decree n.3175 of 18 December 2021 of Italian Ministry of University and Research funded by the European Union – NextGenerationEU; Award Number: Project code CN_00000033, Concession Decree No. 1034 of 17 June 2022 adopted by the Italian Ministry of University and Research, CUPB63C22000650007 Project title “National Biodiversity Future Center - NBFC”. AL received support from Adam Mickiewicz University, Poznań (Poland), funded project 526000/REZ_PROJEKT “Powrót do badań”.
Conceptualization: AL, FG and GM; Methodology, Formal analysis, Investigation, Data Curation on shell and genitalia: FG, DB and GM; Methodology, Formal analysis, Investigation, Data Curation on molecular data: AL, JRP, KS and MP; Writing - Original draft & Writing - Review and Editing: AL, FG and GM; Supervision: FG, AL and GM; Funding Acquisition: AL and GM.
Joanna R. Pieńkowska https://orcid.org/0000-0003-0372-121X
Giuseppe Manganelli https://orcid.org/0000-0002-8453-280X
Małgorzata Proćków https://orcid.org/0000-0003-2240-7306
Debora Barbato https://orcid.org/0000-0003-1105-1711
Katarzyna Sosnowska http://orcid.org/0000-0002-7506-4231
Folco Giusti https://orcid.org/0000-0001-8722-4653
Andrzej Lesicki https://orcid.org/0000-0002-1924-1934
All of the data that support the findings of this study are available in the main text or Supplementary Information.
COI sequences from GenBank used for molecular analysis comparisons (haplotypes in bold)
Data type: docx
16SrDNA sequences from GenBank used for molecular analysis comparisons (haplotypes in bold)
Data type: docx
H3 sequences from GenBank used for molecular analysis comparisons (haplotypes in bold)
Data type: docx
ITS2 sequences from GenBank used for molecular analysis comparisons (haplotypes in bold)
Data type: docx
Concatenated sequences of COI+16SrDNA used in NJ/ML-MEGA7/IQ Tree/RAxML/BI analysis (Fig.
Data type: docx
Explanation note: COI sequences were 615 bp in length. Long 16SrDNA sequences were cut to 829 positions (the alignment of concatenated sequences COI and long 16SrDNA was then 1444 positions in length).
Concatenated sequences of H3 + [(5.8SrDNA)+ITS2+(28SrDNA)] used in NJ/ML-MEGA7/IQ Tree/RAxML/BI analysis (Fig.
Data type: docx
Explanation note: H3 sequences were cut to 279 bp, 5.8SrDNA+ITS2+28SrDNA sequences were 775 positions in length (the alignment of concatenated sequences H3 + [(5.8SrDNA)+ITS2+(28SrDNA)] was therefore 1054 positions).
Concatenated sequences of COI + 16SrDNA long + H3 + [(5.8SrDNA)+ITS2+(28SrDNA)] used in NJ/ML-MEGA7/ML-IQ Tree/RAxML/BI analysis (Fig.
Data type: docx
Explanation note: The lengths of the particular sequences were COI 615 bp, 16SrDNA – 829 bp, H3 – 279 bp, 5.8SrDNA+ITS2+28SrDNA – 775 bp (the alignment of concatenated sequences COI + 16SrDNA long + H3 + [(5.8SrDNA)+ITS2+(28SrDNA)] was therefore 2498 positions).
Maximum Likelihood (ML) tree of 16SrDNA haplotypes of Monacha cantiana
Data type: eps
Explanation note: New 16SrDNA sequences of M. cantiana (Table
Maximum Likelihood (ML) tree of 16SrDNA haplotypes of Monacha cantiana
Data type: eps
Explanation note: New 16SrDNA sequences of M. cantiana (Table
Maximum Likelihood (ML) tree of ITS2 (flanked with 5.8S and 28SrDNA) haplotypes of Monacha cantiana
Data type: eps
Explanation note: New ITS2 sequences of M. cantiana (Table