**1. Introduction**

Arid and semi-arid areas are extremely sensitive to climate change and human activities, and the forest-steppe ecotone in these areas is among the most vulnerable ecosystems in the world [1–3]. Until now, climate change has posed a serious threat to the forest-steppe ecotone in arid and semi-arid areas by causing tree growth decline and tree mortality [2,4,5]. The latest forecasts sugges<sup>t</sup> that the expansion of arid and semi-arid areas will potentially exacerbate regional warming and habitat fragmentation in the future [3,6]. Therefore, there is an urgen<sup>t</sup> need to pay attention to the forest-steppe ecotone in such ecologically fragile areas.

Pollen taxa concentrates in mountains in arid regions, which are also key areas for ecosystem services. Mountain areas typically have rich and unique pollen taxa [7]. Generally, mountain vegetation belts in arid central Asia have various types, ranging from desert to grassland, forest and alpine meadows as the elevation rises [8]. Among them, mountain forest-steppes, typically situated between the forest and steppe belts, are located at the driest edge of forest distribution vertically [9,10]. As such, it is critical to understand the dynamics of the mountain forest-steppe ecotone under climate change in arid regions. Yet, how the change of pollen reflects the migration of the forest-steppe ecotone remains unclear, despite the series of traditional pollen assemblage studies carried out in some hotspots, including the Tibetan Plateau, the Altai Mountains, and the Taibai Mountains [11–13].

Pollen taxa diversity can serve as a comprehensive indicator reflecting the dynamics of the vegetation belts [4]. Whether past climate change increased or decreased pollen taxa diversity has not ye<sup>t</sup> been fully determined, and changes in the pollen taxa diversity potentially include clues for vegetation belt change, especially for the migration of the mountain forest-steppe ecotone. Thus, the Holocene is an ideal period to explore the migration of the forest-steppe ecotone, wherein which climate change was mainly caused by natural variability.

Moreover, it is important to focus on the response modes of mountain vegetation belts to Holocene climate change and to improve the corresponding mechanisms, which are the key to exploring the history of forest-steppe migration. The position of the mountainous vertical vegetation belt is very sensitive to climate change [11,12], because the vertical belt is the epitome of the horizontal belt (about 1/1000). The vertical belt in arid areas is unique, as the upper and lower limits of the forest are a ffected by temperature and moisture, respectively. Mountain vegetation responds to temperature change by position migration, while it responds to moisture change through changes in species composition [4,5]. These two modes are mostly adopted, even though they might not fully explain the mechanism between mountain vegetation belts and changes in temperature and moisture. Studies have indicated that the width of the mountain vegetation belt and climate are not completely in equilibrium [14,15]. As the climate mode can be warm and dry, warm and wet, cold and dry, and cold and wet, both the upper and the lower limits of the forest belt, which are in di fferent climatic conditions, may respond di fferently [12], which ultimately leads to potential changes in the belt width and position. As such, variations in the width of vegetation belts may also be one of the most typical responses to particular climate change, and may further lead to the change in the forest-steppe ecotone. Therefore, it is of importance to see whether these kinds of response patterns exist in the vegetation belts and can be detected through the change of pollen taxa diversity in arid mountain areas.

The Tien Mountains, the largest mountain system in the world's arid regions, is located in the hinterland of Eurasia, a ffected by the westerlies [16–18], where the vertical di fferentiation of vegetation belts is apparent and the typical mountain forest-steppe exists [19]. Due to the complex topographical conditions, limited range size and arid environments, the forest-steppe ecotone in the Tien Mountains appears to be most threatened by climate deterioration. However, few studies have specifically focused on the migration of the forest-steppe ecotone in response to the e ffect of climate change in the Tien Mountains, despite many studies exploring its climate and vegetation changes at multiple time scales [20–23]. Therefore, there is an urgen<sup>t</sup> need to comprehensively study the response pattern of the forest-steppe ecotone in the Tien Mountains to Holocene climate change based on evidence from both surface pollen and fossil pollen, as pollen can provide a long history of pollen taxa diversity with good temporal continuity [4].

Based on the above description, we aim to explore the following scientific questions: (1) How did the mountainous pollen taxa diversity evolve during the Holocene? (2) How did the pollen taxa diversity reflect the changes in vegetation belts in the Tien Mountains during the Holocene? (3) How did the forest-steppe ecotone migrate in response to the Holocene temperature and moisture change in the Tien Mountains?

#### **2. Materials and Methods**

#### *2.1. Study Area*

The Tien Mountains, the largest mountain system in the world's arid regions, are located in the hinterland of Eurasia, influenced by westerlies (Figure 1a,b) [16]. The annual mean precipitation on the northern slopes changes from 175 mm in the desert zone to 465 mm in the mid-altitude zone [24].

**Figure 1.** The location of the Tien Mountains in the hinterland of Eurasia, influenced by westerly winds. (**a**) Digital elevation model image of the study area. The red solid dot represents the location of Sayram Lake, while the blue one represents the location of Aibi Lake. (**b**) The red dotted area represents the range of subgraph. The blue dotted line represents the modern limit of the East Asian summer monsoon, which also illustrates the horizontal limit of the westerlies effect. (**c**) Vegetation belts on the northern slope of the Tien Mountains.

The Tien Mountains have the most typical and complete vertical natural belt spectrum for temperate arid areas around the world, reflecting the distribution characteristics and changes in the mountain pollen taxa diversity and ecological processes. The northern slope of the Tien Mountains can be divided into the following vegetation belts [14] (Figure 1c): sparse and low cushion vegetation is distributed in high mountain belts above 3400 m a.s.l.; alpine and subalpine meadows dominate from 2700 to 3400 m a.s.l.; conifer forest is distributed on the middle elevations from 1700 to 2700 m a.s.l.; typical steppe dominates from 700 to 1700 m a.s.l., represented by steppe on the sunny slope and patchy forest on the shady slope; desert vegetation is distributed below 700 m a.s.l. Notably, the forest-steppe ecotone, also known as the transition zone between the forest belt and the steppe belt, is currently situated around 1700 m a.s.l. on the northern slope of the Tien Mountains [14].

#### *2.2. Modern Vegetation Survey*

To investigate the changes in the richness of modern plant species along the elevational gradient, typical plots representing the characteristic vegetation composition in each vegetation belt were selected, based on the natural geographical pattern of the Tien Mountains [14]. In the mountainous area, with an elevation from 700 to 1700 m a.s.l., plots were sampled every 100 m along the elevational gradient. Due to a flat terrain in the oasis and the downstream desert area, plots were sampled every 50 km in the horizontal direction. Finally, 40 valid plots were surveyed. During the sample survey, the forest plot area was 10 m × 10 m, and the grassland or meadow community area was 2 m × 2 m. Then the detailed information about vegetation habitat conditions, species composition, coverage and height were recorded.

#### *2.3. Surface Pollen*

Surface soil samples were collected every 100 m in the range of 200 to 3500 m a.s.l. on the northern slope of the Tien Mountains, and 34 samples were obtained to investigate the changes in surface pollen composition and evenness in each vegetation belt [14]. Pollen was extracted by treating with HCl (10%) and NaOH (10%), then sieved through 180 μm and 10 μm sieves, and floated with heavy liquid (KI + HI + Zn) using standard techniques [25]. Each sample was counted to contain at least 250 pollen grains under an optical microscope at 400× magnification.

#### *2.4. Fossil Pollen*

We selected two typical fossil pollen sites with high resolution on the northern slope of the Tien Mountains to explore the evolution of pollen taxa diversity. We digitized all pollen taxa using GetData Graph Digitizer v2.25 software [26] and then used them as analytical paleo data. Considering the location representativeness, age, temporal resolution and pollen identification of fossil pollen cores, the following two sites can meet our analysis needs.

The first sediment core, named SLMH-2009 (44◦35 N, 81◦09 E), was from Sayram Lake, with an elevation of 2072 m (Figure 1a,b) [22], and was located in the middle-low part of the modern conifer forest belt. This core was dated back to the Late Glacial to early Holocene transition, with 11 AMS14C dates, and had a temporal resolution of less than 100 years for each pollen sample. The authors analyzed 150 fossil pollen samples in total, with a minimum of 400 pollen grains identified for each sample.

The second sediment core, named A-01, was from Aibi Lake (44◦54–45◦08 N, 82◦35–83◦10 E) (Figure 1a,b) [23], located in the desert vegetation belt at 200 m a.s.l. This core was dated back to the Late Glacial to early Holocene transition with eight AMS14C dates and had a temporal resolution of less than 150 years for each pollen sample. The authors analyzed 195 fossil pollen samples in total, with a minimum of 350 pollen grains identified for each sample.

#### *2.5. Pollen Taxa Diversity Index*

We chose the Shannon-Wiener index and Simpson index to calculate the pollen taxa diversity, as these two indices include the heterogeneity of the measured plant community. They consider both the richness and evenness of the species in the community. Specifically, the Shannon-Wiener diversity index was used to estimate the level of pollen taxa diversity in the modern ecosystem and in the Holocene, which is a comprehensive index reflecting richness and evenness. Its formula is as follows [27].

$$\text{pH} = -\sum\_{i=1}^{r} \text{Pi} \ln \text{Pi} \tag{1}$$

*Pi* = *ni*/*N*, indicating the relative richness of species.

*ni* represents the number of individuals of each pollen taxon.

*N* represents the total number of individuals of all pollen taxa in the community.

It should be noted that communities with low richness and high evenness, and communities with high richness and low evenness, have low diversity indices.

The Simpson diversity index was also used to present the pollen taxa diversity. Unlike the Shannon-Wiener index, the Simpson index measures the probability that two random entities from the pollen group represent di fferent pollen taxa, which can be quantified by the following equation [28].

$$\mathbf{D} = 1 - \sum\_{i=1}^{N} P\_i^2 \tag{2}$$

*Pi* = *ni*/*N*, indicating the relative richness of species. *ni* represents the number of individuals of each pollen taxon. *N* represents the total number of individuals of all pollen taxa in the community.

#### *2.6. Climate Data*

Holocene temperature change was acquired from the integrated results of paleoclimate records for 30–90◦ N [29,30], and Holocene moisture change was derived from integrated results of paleoclimate records in arid central Asia [16].
