Urban structures are poor stepping stones for crevice-nesting ants in fragmented landscapes

D. Satizabal-Niemeyer, Abel Bernadou, C. Wanke, J. Heinze, M. Cordonnier

► To cite this version:

D. Satizabal-Niemeyer, Abel Bernadou, C. Wanke, J. Heinze, M. Cordonnier. Urban structures are poor stepping stones for crevice-nesting ants in fragmented landscapes. Insectes Sociaux, 2024, 72, pp.53-58. 10.1007/s00040-024-01009-7. hal-04828279

HAL Id: hal-04828279

https://hal.science/hal-04828279v1

Submitted on 20 Mar 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Urban structures are poor stepping stones for crevice-nesting ants in fragmented landscapes

D. Satizabal-Niemeyer<fnref n="1" /> · A. Bernadou<fnref n="2" /> Ⓓ · C. Wanke<fnref n="1" /> · J. Heinze<fnref n="1" /> Ⓓ · M. Cordonnier<fnref n="1" /> Ⓓ

Received: 14 November 2024 / Revised: 19 November 2024 / Accepted: 21 November 2024 / Published online: 8 December 2024
© The Author(s) 2024

## Abstract

Despite the high fragmentation of its natural habitats—grasslands with limestone rocks—a previous study revealed that the ant species *Temnothorax nigriceps* show no evidence of genetic isolation between populations separated by kilometers of presumably unsuitable landscape. In the present study, we hypothesized that urban anthropogenic structures might allow the species to nest and maintain efficient gene flow in spite of the ongoing fragmentation. To test this, we surveyed the presence of *T. nigriceps* in many putatively suitable anthropized habitats—such as stone walls, ornamental devices, or urban park structures—in an urban area near a continuous source of propagules from natural habitats. We found very few occurrences of *T. nigriceps* in this urban area, suggesting either a low density of nests in such habitats or difficulty in detecting them in anthropized environment. We discuss the importance of surrounding vegetation and water sources for nesting, especially in anthropized environment where temperatures on mineral surfaces can exceed those in the species’ natural habitats. We conclude that *T. nigriceps* tend to nest in the most natural-like structures, implying that urban habitats provide poor substitute for the species’ native environment.

**Keywords** Ants · Human-mediated dispersal · Nest material · Nest-site selection · Temnothorax species · Urbanization

## Introduction

The ant species *Temnothorax nigriceps* (Mayr 1855) typically inhabits sunny, xerothermic environments with sparse vegetation and bare rocks. Colonies consisting of a few dozen individuals nest in narrow rock crevices (Seifert 2018). Despite the extensive fragmentation of its natural habitat due to anthropogenic activities, such as agriculture or urbanization (Poschlod & Wallis DeVries, 2002; Poschlod et al. 2008), a previous study surprisingly found only limited genetic differentiation among *T. nigriceps* populations (Cordonnier et al. 2022). This pattern could be attributed to either high dispersal capabilities mitigating the negative effects of fragmentation or the species' adaptability to utilize alternative, man-made nesting sites in urban areas. Another

potential explanation is the presence of large effective population sizes. However, the detection of local non-random mating suggests that this hypothesis is less likely (Cordonnier et al. 2022).

Habitat fragmentation is expected to impact *Temnothorax* species even at small spatial scales (e.g., Cordonnier et al. 2023, but see Khimoun et al. 2020). Mating in these small ants is presumably a local event, with nuptial flights occurring over short distances. For example, sexuals of *Temnothorax nylanderi*, a forest-dwelling species, fly within forests or at their edges, avoiding open areas (Noordijk et al. 2008). Though *Temnothorax* sexuals have been recorded more than 100 m above ground and might be occasionally drifted over large areas by wind (Helms et al. 2016), it is unlikely that the number of *T. nigriceps* reproductives traversing large distances is sufficient to maintain gene flow between isolated populations.

The present study explored the hypothesis that *T. nigriceps* utilizes secondary habitats, particularly urban areas, to persist and maintain gene flow despite ongoing anthropogenic fragmentation. Most ant species require minimal resources to establish colonies, suggesting adaptability to various environments. Previous works have shown that *T*.

<figure><img src="image_2.png" /></figure>

<footnote>M. Cordonnier
marion.cordonnier@hotmail.fr</footnote>

<footnote n="1">¹ Chair of Zoology and Evolutionary Biology, University of Regensburg, Regensburg, Germany</footnote>

<footnote n="2">² Centre de Recherches Sur La Cognition Animale, Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, Toulouse, France</footnote>
*nigriceps* readily nests in crevices of man-made stone walls in orchards or vineyards at the edge of villages (Seifert, 2018; Suefuji and Heinze 2014), but the use of such sites in highly modified anthropized environments remains underexplored. For example, *T. nigriceps* might be able to use urban microhabitats, such as rocky elements of urban settlements. The utilization of these artificial structures can provide alternative shelter for wildlife in fragmented environments (Lowry et al. 2013), with some previous evidence also in ants (Cordonnier et al. 2019).

In this study, we investigated the capacity of *T. nigriceps* to utilize man-made structures in urban areas, such as stone walls, masonry, or ornamental structures as alternative nesting sites. This adaptability might facilitate the species' survive in small suitable patches of habitat and provide an explanation for the absence of genetic isolation observed by Cordonnier et al. (2022). If *T. nigriceps* is able to use anthropized habitats, we expect to detect its presence in

urban settlements near its natural habitats, with the latter constituting a continuous source of propagules. To test this hypothesis, various stone structures were examined for the presence of *T. nigriceps* to establish the typology of its anthropogenic habitats.

## Materials and methods

### Study sites and sampling

Fieldwork was conducted in Kallmünz, Germany, a city surrounded by natural *T. nigriceps* habitats where the species is densely present (48 colonies spread all around the city previously collected; green areas in Fig. 1; Cordonnier et al. 2022). The choice of Kallmünz as a study site makes, therefore, particular sense because of the high densities of nests in the direct surroundings (at a few meters from the

<figure><img src="image_3.png" /><figcaption>**Fig. 1** Map of sampling sites in Kallmünz (Germany). Each sampling site corresponds to one suitable mineral element (except sites 1, 2, 3, and 30 with resp. 10, 10, 8, and 15 elements investigated). Yellow: sites investigated in 2022. Blue: sites investigated in 2023. Green areas are natural habitats where *T. nigriceps* is known to occur (based</figcaption></figure>

on Cordonnier et al. 2022) with the number of colonies previously collected (note that this corresponds to approx. 10 colonies sampled per day per person, AB pers. comm.). The sites where *T. nigriceps* has been collected (see “Results” section) are circled in red

<figure><img src="image_4.png" /></figure>
city center), in all directions around the city. No other site
presenting a similar potential exposure (i.e., likely to receive
such a propagule pressure of *T. nigriceps* sexuals) is known.

Within the city, we surveyed all man-made structures
that appeared suitable for the species using the same proto-
col successfully applied in Cordonnier et al. (2022). Urban
mineral elements, such as rocks, bridge fences, and walls
(Figure S1 for exhaustive list) which correspond to potential
nest sites, were carefully inspected by two people for the
presence of nests or ant activity. Surveys were conducted
in early June 2022 (n=29 sites) and July 2023 (n=25 sites;
Fig. 1) at temperatures ranging from 21 to 31 °C. Four sites
were revisited in both years. Each site was mapped and pho-
tographed (Fig. 1, Fig S1). For all potential mineral ele-
ments, the presence of grass and the overall texture of the
mineral surface were recorded. To confirm the occurrence of
the species during the sampling period, one site (number 2,
Fig. 1) within the natural habitat was included and sampled.

In this natural site and in three urban sites (1, 3 and 30),
more than one mineral element was visited (10 elements in
the natural site and 10 elements in site 1, eight elements in
site 3, and 15 elements in site 30, Fig. S2 and S3). In addi-
tion, an alternative protocol using baits (tuna and honey) was
used on the same elements from the sites 1 to 3. Each bait
was left in place for three hours and visited every 10 min the
first hour and then every 20 min the last 2 h. All *Temnotho-*
*rax* ants present on the bait or within a 10 cm radius were
collected for further analysis.

Of the total samples collected for this study, only 10
contained one or more *Temnothorax* ants (seven samples in
2022, three samples in 2023). None of these records resulted
from baiting; all were obtained through direct searches on
mineral surfaces, using the same method as in Cordonnier
et al. (2022). This suggests that baiting is not an effective
sampling method for detecting slow-moving species of the
*Temnothorax* genus.

Morphological and genetic identification

Morphological characteristics of all *Temnothorax* individu-
als (minimum five when more individuals were collected
per collection site) were examined using a Leica binocular
microscope (magnification: 30x). Particular attention was
given to femora coloration (central portions darkened in *T.
nigriceps*), overall body color (brown to almost black), and
head-mesosoma contrast (head distinctly darker than meso-
soma) (Salata & Borowiec 2013).

To confirm the identity of the species, genomic DNA was
extracted from a single worker per sample (n=10) using a
CTAB method (modified from Sambrook & Russell, 2001).
All samples were Sanger sequenced for a stretch covering
the mitochondrial genes CO I and II including tLeu (primers
C1-J-2183 Jerry: 5'-CAACATTTATTTTGATTTTTTGG-3'

/ Cw.3031: 5´-TTTGCMCTWATCTGCCMTATT-3´). The 25 µl PCR reaction volume consisted of 12.5 µl buffer with Taq DNA polymerase, 9.5 µl ddH2O, 1 µl reverse primer, 1 µl forward primer (final concentration of 0.5 µM) and 1 µl DNA (2–10 ng). PCR consisted of initial denaturation at 94 °C (4 min), 37 cycles at 94 °C (denaturation, 45 s), 50 °C (annealing, 45 s) and 72 °C (elongation, 60 s), and a final step at 72 °C (5 min). PCR products were cleaned up using the NucleoSpin Gel and PCR Macherey-Nagel's Clean-up Kit and sequencing was conducted by LCG Genomics GmbH (Berlin, Germany).

Phylogenetic relationships among the mitochondrial hap-
lotypes were based on sequence fragments ranging from
799 to 1409 bp in length (Genebank accession numbers:
[PQ672614-PQ672622]). The sequences generated in this
study were compared with sequences obtained from Gen-
Bank, including 9 *Temnothorax* species present in Ger-
many and 2 *Leptothorax* species, covering all morphologi-
cally identified species except *T. albipennis*, for which no
sequence was available. *T. albipennis*, along with *T. nigri-
ceps* (Mayr), *T. tuberum* (F.), and *T. unifasciatus* (Latr.)
forms a group of closely related species with tendencies for
hybridization, often co-occurring in the same habitat (Dou-
wes & Stille 1991, Salata & Borowiec 2013). Sequences
were aligned using default options in MUSCLE v3.8.31
(Edgar 2004) as implemented in SeaView v4.2.9 (Gouy et al.
2010). The relationships were evaluated based on a tree con-
structed using the PhyML algorithm with the GTR distance
without invariable sites, optimized nucleotide equilibrium
frequencies, and tree-searching operations involving best of
NNI and SPR.

Results

Among the 10 samples analyzed, five were identified as *T. nigriceps* based on morphological traits, which were subsequently confirmed by their mitochondrial DNA sequences (Fig. 2). This included one individual collected from the species' natural habitat (sampling site 2 in Fig. 1). *T. nigriceps* was thus sampled in only four human-made or human-modified environments (two samples in site 3, one sample from site 5 and one sample from site 6, Figs. 1 and 3). The five other samples belonged to *T. interruptus* (site 4), *Leptothorax gredleri* (site 8), *L. acervorum* (site 30), and unidentified *Temnothorax* species (sites 7 and 30—morphologically similar to *T. albipennis*). As a result, over the 3 days of sampling, the targeted species was recorded only four times, indicating either a low density of nests in urban habitats and/or a relatively hard detection in anthropized environments.

Among the suitable mineral elements investigated, those
where *T. nigriceps* was collected probably were most similar
to the species' natural habitat (see example in site 2 picture,
Sequencing

PhyML ln(L)=-4847.5 1573 sites GTR 4 rate classes
0.1

Morphology

<figure><img src="image_5.png" /><figcaption>**Fig. 2** Tree obtained from the COI-COI sequences built using the PhyML algorithm with the GTR distance without invariable sites, optimized nucleotide equilibrium frequencies, and tree-searching operations involving best of NNI and SPR. For all sequences generated in this study, the associated morphological identification is indicated in green. All other sequences are named with the species ID and their GenBank accession number</figcaption></figure>

Fig. 3). These collection sites, except for site 6 (Fig. 3), typi-
cally were older, unprocessed stones with rough, porous sur-
faces and associated vegetation. Two samples were collected
within 50 m of each other at site 3 (graveyard of the city), in
the same type of nesting material (Fig. 3).

**Discussion**

The rare detection of *T. nigriceps* suggests that it is absent
from the urban area. The species may be highly selective in
its habitat requirements and, therefore, may avoid nesting
in highly transformed mineral surfaces. This result could
be extended to other species within the genus as none of
the records of *Temnothorax* or *Leptothorax* sp. was from
material processed by humans. In addition, the presence of
competing ant species in urban areas, often characterized by
aggressive behavior and larger colony sizes, may hinder the
establishment of *Temnothorax* colonies through competition
by interference or by direct interaction (Gibb and Hochuli
2003; Holway and Suarez 2006; Stringer et al. 2009).

*Temnothorax* species are generally considered predators
or scavengers (Seifert 2018; Giannetti et al. 2022), but
some of them can also feed on dropped-off honey dew,
nectar of floor plants or crushed nuts (Fokuhlet al. 2012).

In addition, species, such as *T. nylanderi*, exhibit a limited
foraging range around their nest (Heinze et al., 1996). *T.
nigriceps*, therefore, likely depends on microhabitats offer-
ing sufficient prey or alternative food sources. While the
presence of suitable nesting sites is essential, the avail-
ability of adequate food resources is another critical factor
for colony establishment and persistence in urban areas.

The low number of *T. nigriceps* samples collected in
urban area during the study period may also be partly
attributed to a low detection of the nests rather than an
absence of the species. The relatively small colony size
and cryptic behavior typical of *Temnothorax* and *Lepto-
thorax* species likely contribute to their low detectability
in complex urban environments. In addition, high ambient
temperatures (> 30 °C) may have reduced ant activity on
overheated mineral substrates. Urban areas tend to favor
ant species adapted to warmer, drier environments (Menke
et al. 2011), which could influence the distribution of *T.
nigriceps* within the city. The possibility of seasonal nest
displacement in microhabitats with cooler conditions can-
not be excluded. Earlier sampling, such as in early May,
might have yielded higher detection rates. However, the
detection of the species in its natural habitat under similar
high-temperature conditions suggest that the low presence

<figure><img src="image_6.png" /></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 3** Pictures of the collection sites where *Temnothorax* or *Lepthothorax* individuals have been collected. Site 2 (top left) is a rock located in natural habitat of *T. nigriceps*. All the other mineral elements</figcaption></figure>

are located in the urban area of Kallmünz, Germany (see Fig. 1 for the map). The species identified based on mtDNA (or morphology for *T. albipennis*) are indicated below each picture

of *T. nigriceps* in urban areas is primarily linked to its
limited occupation of anthropogenic structures.

Consistent with the previous factors (i.e., urban areas
providing drier and warmer shelter, and necessity to find
food resources around the suitable nest material), cem-
eteries could represent refuges for *T. nigriceps*, offering a
combination of nesting substrates, vegetation, and water
resources. These conditions, combined with reduced
human disturbance, may create favorable habitats for both
the ants and their nutritional resources. The role of cem-
eteries in maintaining biodiversity within cities is known
for a large range of taxa (Löki et al. 2019), with previous
examples in ants (e.g., Klesniaková et al. 2016).

Based on our findings, urban habitats appear to be poor
substitutes for the natural habitats of *Temnothorax nigri-
ceps*. A wide utilization of human-modified mineral ele-
ments as alternative shelters to prevent isolation and main-
tain the gene flow between fragmented populations of *T.
nigriceps* is, therefore, unlikely. Punctual human-mediated
dispersal by translocation of nesting material from natural
habitats, or the use of other, right now unidentified types
of habitats would be forthcoming more efficient to prevent
the genetic isolation of the population.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s00040-024-01009-7.

**Acknowledgements** We thank E. Eichelbrönner and M.-T. Cordonnier for the help in collecting data. We thank A. Trindl for the help with molecular ecology.

**Author contributions** Conceptualization: Marion Cordonnier; methodology: Marion Cordonnier; formal analysis and investigation: Daniel Satizabal-Niemeyer, Christiane Wanke, Abel Bernadou, Marion Cordonnier; writing—original draft preparation: Daniel Satizabal-Niemeyer, Marion Cordonnier; writing—review and editing: Abel Bernadou, Jürgen Heinze, Marion Cordonnier; funding acquisition: Marion Cordonnier; supervision: Jürgen Heinze.

**Funding** Open Access funding enabled and organized by Projekt DEAL. A.B. was supported by the Centre National de la Recherche Scientifique, University Paul Sabatier, and the Agence Nationale de la Recherche (grant ANR-22-CPJ2-0133-01). M.C. was supported by an Alexander von Humboldt postdoctoral fellowship.

**Data availability** The sequences generated in this study are available on Genebank (accession numbers: [PQ672614-PQ672622]).

**Declarations**

**Conflicts of interests** The authors have no competing interests to declare that are relevant to the content of this article.
**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

## References

Cordonnier M, Gibert C, Bellec A, Kaufmann B, Escarguel G (2019) Multi-scale impacts of urbanization on species distribution within the genus Tetramorium. Landscape Ecol 34:1937–1948

Cordonnier M, Felten D, Trindl A, Heinze J, Bernadou A (2022) Absence of genetic isolation across highly fragmented landscape in the ant *Temnothorax nigriceps*. BMC Ecology and Evolution 22(1):1–11

Cordonnier M, Lindner T, Heinze J (2023) Fragmentation shapes nest density and social structure but not genetic diversity of *Temnothorax crassispinus* (Formicidae). Popul Ecol 65(3):192–202

Douwes P, Stille B (1991) Hybridization and variation in the Leptothorax tuberum group (Hymenoptera: Formicidae). J Zool Syst Evol Res 29(3):165–175

Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32(5):1792–1797

Fokuhl G, Heinze J, Poschlod P (2012) Myrmecochory by small ants—Beneficial effects through elaiosome nutrition and seed dispersal. Acta Oecologica 38:71–76

Giannetti D, Schifani E, Gugliuzzo A, Zappalà L, Biondi A, Grasso DA (2022) Native European ants can discourage host colonization and reduce reproductive success of the invasive ambrosia beetle Xylosandrus compactus. Biol Control 174:105032

Gibb H, Hochuli DF (2003) Colonisation by a dominant ant facilitated by anthropogenic disturbance: effects on ant assemblage composition, biomass and resource use. Oikos 103(3):469–478

Gouy M, Guindon S, Gascuel O (2010) SeaView version 4: a multi-platform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol 27(2):221–224

Heinze J, Foitzik S, Hippert A, Hölldobler B (1996) Apparent enemy phenomenon and environment-based recognition cues in the ant *Leptothorax nylanderi*. Ethology 102(3):510–522

Helms JA, Godfrey AP, Ames T, Bridge ES (2016) Predator foraging altitudes reveal the structure of aerial insect communities. Sci Rep 6(1):28670

Holway DA, Suarez AV (2006) Homogenization of ant communities in Mediterranean California: the effects of urbanization and invasion. Biol Cons 127(3):319–326

Khimoun A, Doums C, Molet M, Kaufmann B, Peronnet R, Eyer PA, Mona S (2020) Urbanization without isolation: the absence of genetic structure among cities and forests in the tiny acorn ant *Temnothorax nylanderi*. Biol Lett 16(1):20190741

Klesniaková M, Holecová M, Pavlíková A (2016) Interesting ant species in the urban greenery of Bratislava. Folia faunistica Slovaca 21(3):235–238

Löki V, Deák B, Lukács AB, Molnár A (2019) Biodiversity potential of burial places—a review on the flora and fauna of cemeteries and churchyards. Global Ecology and Conservation 18:e00614

Lowry H, Lill A, Wong BB (2013) Behavioural responses of wildlife to urban environments. Biol Rev 88(3):537–549

Menke SB, Guénard B, Sexton JO, Weiser MD, Dunn RR, Silverman J (2011) Urban areas may serve as habitat and corridors for dry-adapted, heat tolerant species; an example from ants. Urban Ecosystems 14:135–163

Noordijk J, Morssinkhof R, Boer P, Schaffers AP, Heijerman T, Sýkora KV (2008) How ants find each other; temporal and spatial patterns in nuptial flights. Insectes Soc 55:266–273

Poschlod P, WallisDeVries MF (2002) The historical and socioeconomic perspective of calcareous grasslands—lessons from the distant and recent past. Biol Cons 104(3):361–376

Poschlod P, Karlík P, Baumann A, Wiedmann B (2008) The history of dry calcareous grasslands near Kallmünz (Bavaria) reconstructed by the application of palaeoecological, historical and recent-ecological methods. Human nature: studies in historical ecology and environmental history. Institute of Botany of the Czech Academy of Sciences, Brno, CZ, 130–143.

Salata S, Borowiec L (2013) *Temnothorax albipennis* (CURTIS, 1854) in Poland and identification of the *T. tuberum* species complex (Hymenoptera: Formicidae). Genus 24(3–4):403–413

Sambrook J, Russell DW (2001) Detection of DNA in agarose gels. Molecular Cloning, A Laboratory Manual, (3rd Ed.) Cold Spring Harbor Laboratory Press, New York, 5–14.

Seifert B (2018) The ants of Central and North Europe. Lutra Verlags- und Vertriebsgesellschaft, Tauer, Germany, p 408

Stringer LD, Stephens AE, Suckling DM, Charles JG (2009) Ant dominance in urban areas. Urban Ecosystems 12:503–514

Suefuji M, Heinze J (2014) The genetic population structure of two socially parasitic ants: “the active slave-maker” *Myrmoxenus ravouxi* and the “degenerate slave-maker” *M. kraussei*. Conserv Genet 15:201–211

<figure><img src="image_8.png" /></figure>